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Multiple colonizations lead to cryptic biodiversity in an island ecosystem: comparative phylogeography of anchialine shrimp species in the Ryukyu Archipelago, Japan.


Geological events (tectonics, volcanism, sea-level changes, etc.) can significantly impact the flora and fauna of an area, consequently shaping the structure and evolutionary trajectory of populations (e.g., Vandergast et al., 2004; Liggins et al., 2008; Santos and Weese, 2011). In this respect, oceanic islands, with their discrete geologic history and generally high levels of endemism, are ideal systems for studying how such events influence organismal diversification and community composition. Archipelagos of the Indo-West Pacific have received considerable interest in this regard (Liu, X. et al., 2008) since the species diversity of many of these island groups is among the richest in the world (Myers et al., 2000: Meijaard, 2003). Although a number of hypotheses have been proposed to explain the high species diversity of these island groups (reviewed by Carpenter et al., 2011), phylogeographic studies generally support one of two alternatives: (i) the center of origin hypothesis, in which the majority of the region's biodiversity is endemic (i.e., due to repeated bouts of isolation by geologic events) and a source of species into surrounding areas (Benzie, 1998; Briggs, 2000; Barber et al., 2006); or (ii) the center of accumulation hypothesis, whereby this high biodiversity is due to species accumulation via dispersal from surrounding areas (i.e., Indian, Pacific, or Australasian biotas; Murphy and Austin, 2005; Page et al., 2007; De Bruyn and Mather, 2007). However, with a few exceptions (see Bird et al., 2007, 2011), many studies conducted to date have focused on single species, often leading to an inability to differentiate between these two possibilities (see De Bruyn and Mather, 2007).

The Ryukyu Archipelago, located in the Indo-West Pacific and stretching between Japan and Taiwan, has experienced an extensive range of geological events over its history. Specifically, the backbone of this island chain formed during the late Miocene (~10 MYA) after subduction of the Philippine Plate by the Eurasian Plate (Konishi and Sudo, 1972; Koba, 1980; Lee et al., 1980). During low sea-level stands associated with glacial periods of the late Pliocene (~2.5-3 MYA), a landbridge is thought to have extended to southeastern China, connecting the central Ryukyus with Taiwan and continental Asia (Kimura, 2000). Furthermore, this landbridge was submerged during the interglacial periods of the early Pleistocene (~2-2.5 MYA), creating islands corresponding to the present island groups (Hikida and Motokawa, 1999); these experienced sequential periods of connection and isolation due to fluctuating sea levels throughout the Pleistocene (Lin et al., 2002). These latter events are believed to have significantly influenced the distributions and diversification of fauna in the Ryukyus, and the phylogenetic relationships of several terrestrial taxa reflect these paleogeogaphic patterns (Suzuki, 2001). For example, Lin et al. (2002) found that the phylogeny of Takydromus grass lizards strongly correlated with the sequential separation of islands during the late Pleistocene. Similar patterns have also been reported for Salganea and Panesthia wood-feeding cockroaches (Maekawa et al., 1999), Parachauliodes fishflies (Liu. X. et al., 2008), and Anomala beetles (Muraji et al., 2008).

Another group whose evolutionary history is predicted to correlate with the geologic history of the Ryukyu Archipelago is organisms from the anchialine niche. This ecosystem comprises land-locked bodies of salt or brackish water that fluctuate with the tides due to subterranean connections to both the open ocean and the freshwater aquifer systems (Holthuis, 1973; Maciolek, 1983). Such habitats have been reported from the Sinai Peninsula, Bermuda, the Caribbean, the Hawaiian Islands, the South Pacific, and the Philippines, as well as the Ryukyus (Maciolek, 1986). While exceptions exist (Kano and Kase, 2004; Russ et al., 2010; Cabezas et al., 2012), numerous genetic studies have revealed cryptic species complexes with exceptional levels of endemism and strong population structure on the scale of only a few kilometers for a number of anchialine organisms (Santos, 2006; Craft et al., 2008; Hunter et al., 2008; Page et al., 2008; Santos and Weese, 2011; Weese et al., 2012). For example. populations of the anchialine shrimp Caridina rubella Fujin and Shokita, 1975 (Decapoda: Atyidae) on the island of Miyako in the Ryukyus likely belong to two distinct species that are significantly structured across distances ranging from less than 20 m to more than 10 km (Weese et al., 2012). Additionally, anchialine habitats have generally been heavily influenced by sea-level changes during the Quaternary (Mylroie and Mylroie, 2011); for an-chialine organisms in the Ryukyus, such migrating coastlines may have led to multiple cycles of contraction and expansion of suitable habitats (i.e., the subterranean aquifer system) in the archipelago. In this context, comparing the phylogeography of different taxa from this ecosystem provides an opportunity to understand how past environmental changes may have impacted patterns of anchialine biodiversity in the Ryukyus and the species diversity of Indo-West Pacific archipelagos in general.

Decapod crustaceans are the dominant macrobiota of the anchialine ecosystem in the Ryukyus, with at least 11 shrimp and crab species having been recorded (Komai and Fujita, 2005; Cai and Shokita, 2006; Naruse and Tamura, 2006; Fujita, 2007; Fujita and Sunagawa, 2008). Of these, three species of caridean shrimp--Antecaridina lauensis Edmondson, 1935 (Decapoda: Atyidae), Halocaridinides trigonophthalma Fujino and Shokita, 1975 (Decapoda: Atyidae), and Metabetaeus minutus Whitelegge, 1897 (De-capoda: Alpheidae)--have disjunct distributions in the Ryukyus as well as across the Pacific Basin (Cai and Shokita, 2006; Anker, 2010). If, as suggested by the center of origin hypothesis, the dynamic geologic history of the Ryukyus contributed to the distributions and diversification of anchialine diversity on these islands, it is hypothesized that the phylogeographic patterns and evolutionary relationships of A. lauensis, H. trigonophthalma, and M. minutus in the archipelago would reflect these events. On the other hand, if the phylogeographic patterns and evolutionary relationships of these species are not correlated with the sequential separation of island groups in the Ryukyus, this would imply that the anchialine diversity of the archipelago is due to the accumulation of distinct lineages from elsewhere via dispersal (i.e., the center of accumulation hypothesis). To differentiate between these two alternatives, the genetic diversity, phylogeography, and evolutionary history of the three species were investigated across the Ryukyus. Specifically, sequence analyses of the mitochondrial (mtDNA) cytochrome c oxidase subunit I [C0I] gene were employed to measure genetic variation and estimate population structure. Sequences from the mtDNA large ribosomal subunit (16S-rDNA) were then utilized to infer phylogenetic relationships among A. lauensis, H. trigonophthalma, arid M. minutus from within and outside the Ryukyus. Using this combined approach, this study illuminates the complex biogeography of these three endemic anchialine caridean shrimp species in the Indo-West Pacific.

Materials and Methods

Taxon sampling and DNA extraction and sequencing

About 42 anchialine habitats were surveyed from nine islands belonging to the Daito, Miyako, and Yaeyama island groups of the Ryukyu Archipelago (Fig. 1) between January and August 2009. From these, specimens of Antecaridina lauensis, Halocaridinides trigonophthalma, and Metabetaeus minutus were acquired from 11 anchialine habitats spanning the six islands of Ishigaki, Taketomi, Tarama, Miyako, Irabu, and Minami-daito (Fig. 1). From each habitat, 6-17 individuals (Table 1) were collected by using baited traps or small aquarium nets and immediately preserved in >90% ethanol for genetic analyses. For phylogenetic analyses (see below), an individual representing "short rostrum" Caridina rubella (see Weese et al., 2012) was collected from Miyako, and additional Antecaridina and Metabetaeus specimens from outside the Ryukyus were supplied by Dr. Arthur Anker (Florida Museum of Natural History) as materials preserved in 75% ethanol.

Table 1

Sample sizes and indices of genetic diversity for population
s of Antecaridina buensis, Halocaridinides trigonophthalma, and
Metahetaeus minutus from the Ryukyu Archipelago, Japan. based
on cviochrome c oxidise subunit I (COI) sequences

Island        Site Name                  Antecaridina
(code)        (code)                         lauensis

                           n  nh   [pi]             h   n  nh   [pi]

Takemmi (TK)  Mi-na-ga                                 16  14  0.028

              Yoshino     16   7  0.065         0.775   6   3  0.001

Turama (TA)   Shuga-ga    17  10  0.003         0.838


              Fushato-ga                               16  14  0.005

Miyako (MY)   Kikya-ga

              Tomori                                    8   4  0.002

Irabu (IR)    Sabauki                                  15   4  0.001
              Well (SW)|

Minami-daito  Jr. High    15   9  0.004         0.876
(MD)          Cave (JR)

              Shiutou     10   7  0.004         0.876

              Cave (MIY)

              Total       58  27  0.040         0.868  61  33  0.094

Island        Site Name   Halocaridinides                 Metabetaeus
(code)        (code)      trigonophthalma                     minutus

                                        h   n  nh   [pi]            h

Takemmi (TK)  Mi-na-ga              0.983

              Yoshino               0.600   6   6  0.006        1.000

Turama (TA)   Shuga-ga

              Futatsu-ga                    6   5  0.007        0.933

              Fushato-ga            0.975

Miyako (MY)   Kikya-ga                      9   8  0.005        0.972

              Tomori                0.643

Irabu (IR)    Sabauki               0.371
              Well (SW)|

Minami-daito  Jr. High                      6   5  0.006        0.933
(MD)          Cave (JR)


              Miyahira                      6   6  0.006         1.00
              Cave (MIY)

              Total                 0.878  33  23  0.051        0.966

n, number of sampled individuals; nh, number of unique
haplotypes; [pi], nucleotide diversity; h, haplotype diversity.

Total genomic DNA was extracted from each individual using 2X cetyltrimethyl ammonium bromide (CTAB)/chloroform according to procedures in Santos (2006). Between 10 and 30 ng of DNA was utilized as template to amplify an ~670 base pair (bp) fragment of the mitochondrial (mtDNA) cytochrome c oxidase subunit I (COI) gene via polymerase chain reaction (PCR). Reactions were ducted in 25-[micro]l volumes containing 10 mmol [1.sup.-1] Tris-HC1 (pH 8.3), 50 mmol [1.sup.-1] KCI, 0.001% gelatin, 2.0 mmol [1.sup.-1] Mg[Cl.sub.2], 200 [micro]mol [1.sup.-1] dNTPs, 0.4 [micro]mol I[1.sup.-1] each of primers LC01490 and HCO2198 (Folmer et al., 1994), and 1 U Taq polymerase. Reactions were performed in a PTC-100 ther-mocycler (MJ Research, Watertown, MA) using the cycling profile from Santos (2006). Additionally, sequence data from the mtDNA large ribosomal subunit (16S-rDNA) gene were obtained from one to two individuals for each divergent genetic lineage within the three "species" (see below) for phylogenetic analyses. The PCRs for an ~850-bp fragment of the 16S-rDNA were conducted in 25-[micro]l volumes with the "touchdown" thermocycling profile outlined in Craft et al. (2008) and containing 0.4 [micro]mol 1 [1.sup.-1] each of primers CRUST16SF and CRUST16SR (Ivey and Santos, 2007) along with the reaction constituents outlined above.

Amplicons were purified with Montage PCR filter units (Millipore, Billerica, MA) according to the supplier's directions, cycle-sequenced in both directions using Big-Dye Terminators ver. 3.1, and read on a PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Ambiguities in chromatograms were corrected by comparison with the complementary DNA strand in SEQUENCHER ver. 4.7 (Gene Codes Corporation, Ann Arbor, MI). Finished COI and 16S-rDNA sequences were aligned manually using SE-AL ver. 2.0all (Rambaut, 2008).

Genetic diversity and population genetic analyses

Population-level analyses utilized mtDNA COI sequence data since this gene has proven informative for other atyid and alpheid species (Cook et al., 2006; Page et al., 2007; Russ et al., 2010). Nucleotide ([pi]) and haplotype (h) diversity estimates were calculated according to the methods of Nei (1987) using DnaSP ver. 5.10.00 (Rozas et al., 2003). To assess potential genetic differentiation between populations, pairwise ([[PHI].sub.ST] statistics (which incorporate information from both haplotype frequencies and molecular divergence) were calculated with Arlequin ver. 3.11 (Excoffier et al., 2005). For these comparisons, the Tamura and Nei (1993) model of DNA evolution, as selected by the Akaike Information Criterion (AIC) with the FindModel web version of Modeltest (Posada and Crandall, 1998: Posada, 2006), was employed. As another measure of potential genetic differentiation, Hudson's (2000) nearest-neighbor statistic ([S.sub.nn]), which measures how often the "nearest neighbors" (in sequence space) are from the same locality, was calculated using DnaSP.

To visualize relationships among COI haplotypes, networks were constructed via TCS ver. 1.21 (Clement et al., 2000), which utilizes the cladogram estimation algorithm of Templeton et al. (1992). These analyses were conducted under default settings, providing 95% parsimony plausible branch connections between haplotypes. Reticulations in networks, representing ambiguous connections, were resolved using the criteria outlined in Crandall et al. (1994). In cases where multiple networks were recovered per species, the historical demography of populations in each network was inferred using Tajima's D (Tajima, 1989) and Fu's [F.sub.s] (Fu, 1997) neutrality tests conducted in Arlequin. Both methods provide information on demographic history in the absence of selection, with significant negative or positive values generally suggesting population expansions or bottlenecks (Tajima, 1989; Fu, 1997; Akey et al., 2004), respectively. Statistical significance in the pairwise [[PHI].sub.ST] and neutrality tests was assessed in Arlequin, as well as the [S.sub.nn] in DnaSP, by 1000 permutations.

Phylogenetic analyses

Phylogenetic analyses utilizing mtDNA 16S-rDNA sequence data were used to infer evolutionary relationships among divergent genetic lineages recovered from the three shrimp species (see Results) examined in this study. For phylogenetic analyses, two datasets were assembled and analyzed in the same manner. The first consisted of atyid 16S-rDNA sequences, including representatives of the A. lauensis, H. trigonophthalma, and "short rostrum" Caridina rubella sequences generated here along with additional Antecaridina spp. and Halocaridina rubra Holthuis, 1973 (Decapoda: Atyidae) sequences from GenBank (Table 2). The second dataset comprised alpheid 16S-rDNA sequences, including representatives of M. minutus, M. lohena Banner and Banner. 1960 (Decapoda: Alpheidae), and M. mcphersonae Anker, 2010 (Decapoda: Alpheidae) from this study and additional Metabetaeus sp. and Betaeus harrimani Rathbun. 1904 (Decapoda: Alpheidae) sequences acquired from GenBank (Table 2). Both datasets utilized Macrob rachium japonicum De Haan, 1849 (Decapoda: Palaemonidae) as the outgroup. The 16S-rDNA sequences were aligned using ClustalX (Thompson, 1997) and adjusted manually using SE-AL. For each dataset, evolutionary relationships were inferred via Maximum Likelihood (ML) analyses with PHYML ver. 3.0 (Guindon and Gascuel, 2003) under the appropriate model of evolution chosen by the AIC in Modeltest ver. 3.7 (Posada and Crandall. 1998). For each phylogeny, the transition/transversion ratio and proportion of invariable sites were estimated, with the starting tree determined by BioNJ (default settings). Branch supports were estimated by 1000 bootstrap replicates and resulting phylogenetic trees visualized with FigTree ver. 1.3.1 (Rambaut. 2009).

Table 2

Results of neutrality tests for genetic lineages of Antecaridina
lauensis. Halocaridinides trigonophthalma, and Metabetaeus
minutus front the Ryukyu Archipelago, Japan, based on
cytochrome c oxidase subunit I (COI) sequences

Species          Lineage             n  nh        Fu's   Tajima's
                                             [F.sub.s]          D

Antercaridina    Ishigaki           10   4    -1.345 *  - 1.667 *

                 Ishigaki/Tarama    48  23  -19.819 **  -1.951 **
                 /Minami- daito

Halocaridinides  Ishigaki/Miyako    24   9   -6.021 **  -2.021 **
trigonophthalma  /Irabu

                 Tarama/Taketomi    30  24  -22.944 **    - 2.024

                 Taketomi            2   1          NA         NA

Metabetaeus      Minami-daito       12   9      -2.650      0.412

                 Miyako/Tarama/     21  14   -7.328 **   -1.842 *

n, number of sampled individuals; nh, number of unique
haplotypes: NA, not attempted.

* P < 0.05, ** P < 0.001.


Genetic diversity

Direct sequencing of mtDNA COI amplicons yielded a 630-bp gene fragment from each of the 33, 61, and 58 individuals of Antecaridina lauensis, Halocaridinides trigonophthalma, and Metabetaeus minutus analyzed in this study. From these, 27 A. lauensis, 33 H. trigonophthalma, and 23 M. minutus, unique COI haplotypes were identified (Table 1, Table A1 [see appendix]) and deposited into GenBank under accession numbers KC879738--KC879820. Although nuclear copies of mitochondrial derived genes (numts: Lopez et al., 1994) can be common and potentially problematic in arthropods (Buhay, 2009), translation of these COI nucleotide sequences into amino acids found no stop codons, and any identified non-synonymous changes were to amino acid residues with similar biochemical properties (data not shown). This finding implies that sequences analyzed here were derived from mitochondrial copies, rather than numts, of COI.

Table A1 Distribution of cariden cytochrome c oxidase sybunit
I (COI) haplotpes for population of Antecaridina lauensis,
Halocaridinides trigono-phthalma, and Metabetaeus minutus
from the Ryukyus Archipelago, Japan

Haplotype     Genebank                                   Sampling

                         MIN  YOSII  SHUG           FUT      FUSH


A1_IS1        KC879738            7

A1_IS2        KC879739            1

A1_IS3        KC879740            1

A1_IS4        KC879741            1

A1_IS5        KC879742            4     7

A1_IS6        KC879743            1

A1_IS7        KC879744            1

A1_MD1        KC879745

A1_MD2        KC879746

A1_MD3        KC879747

A1_MD4        KC879748                  1

A1_MD5        KC879749

A1_MD6        KC879750

A1_MD7        KC879751

A1_MD8        KC879752

A1_MD9        KCK79753                  1

A1_MD10       KC879754

A1_MD11       KC879755

A1_MD12       KC879756

A1_MD13       KC879757

A1_TA1        KC879758                  1

A1_TA2        KC879759                  1

A1_TA3        KC879760                  1

A1_TA4        KC879761                  1

A1_TA5        KC879762                  2

A1_TA6        KC879763                  1

A1_TA7        KC879764                  1

Individuals/                     16    17

Ht_TK1        KC879765     1

Ht_TK2        KC879766     1

Ht_TK3        KC879767     2

Ht_TK4        KC879768     1

Ht_TK5        KC879769     1                                    3

Ht_TK6        KC879770     1

Ht_TK7        KC879771     1

Ht_TK8        KC879772     1

Ht_TK9        KC879773     1

Ht_TK10       KC879774     1                                    1

Ht_TK11       KC879775     1                                    1

Ht_TK12       KC879776     1                                    1

Ht_TK13       KC879777     1

Ht_TK14       KC879778     1

Ht_TA1        KC879779                                          1

Ht_TA2        KC879780                                          1

Ht_TA3        KC879781                                          1

Ht_TA4        KC879782                                          1

Ht_TA5        KC879783                                          1

Ht_TA6        KC879784                                          1

Ht_TA7        KC879785                                          1

Ht_TA8        KC879786                                          1

Ht_TA9        KC879787                                          1

Ht_TA10       KC879788                                          1

Ht_MY1        KC879789

Ht_MY2        KC879790            4

Ht_MY3        KC879791

Ht_MY4        KC879792            1

Ht_MY5        KC879793            1

Ht_MY6        KC879794

Ht_MY7        KC879795

Ht_MY8        KC879796

Ht_MY9        KC879797

Individuals/              16      6                            16

Mm_IS1        KC879787            1                   1

Mm_IS2        KC879799            1

Mm_IS3        KC879800            1

Mm_IS4        KC879801            1

Mm_TA1        KC879802                                1

Mm_TA2        KC879803                                1

Mm_TA3        KC879804                                1

Mm_MY1        KC879805

Mm_MY2        KC879806

Mm_MY3        KC879807

Mm_MY4        KC879808

Mra_MY5       KC879809

Mm_MY6        KC879810            1

Mm_MY7        KC879811            1                   2

Mm_MD1        KC879812

Mm_MD2        KC879813

Mm_MD3        KC879814

Mm_MD4        KC879815

Mm_MD5        KC879816

Mm_MD6        KC879817

Mm_MD7        KC879818

Mm_MD8        KC879819

Mm_MD9        KC879820

Individuals/                      6                   6

Haplotype     Genebank   Localities

                                KIK  TAG  SW  JR  SHIN  MTY

A1_IS1        KC879738

A1_IS2        KC879739

A1_IS3        KC879740

A1_IS4        KC879741

A1_IS5        KC879742                         5     4

A1_IS6        KC879743

A1_IS7        KC879744

A1_MD1        KC879745                         3

A1_MD2        KC879746                         1

A1_MD3        KC879747                         1

A1_MD4        KC879748                         1

A1_MD5        KC879749                         1

A1_MD6        KC879750                         1

A1_MD7        KC879751                         1

A1_MD8        KC879752                         1     1

A1_MD9        KCK79753                               1

A1_MD10       KC879754                               1

A1_MD11       KC879755                               1

A1_MD12       KC879756                               1

A1_MD13       KC879757                               1

A1_TA1        KC879758

A1_TA2        KC879759

A1_TA3        KC879760

A1_TA4        KC879761

A1_TA5        KC879762

A1_TA6        KC879763

A1_TA7        KC879764

Individuals/                                  15    10

Ht_TK1        KC879765

Ht_TK2        KC879766

Ht_TK3        KC879767

Ht_TK4        KC879768

Ht_TK5        KC879769

Ht_TK6        KC879770

Ht_TK7        KC879771

Ht_TK8        KC879772

Ht_TK9        KC879773

Ht_TK10       KC879774

Ht_TK11       KC879775

Ht_TK12       KC879776

Ht_TK13       KC879777

Ht_TK14       KC879778

Ht_TA1        KC879779

Ht_TA2        KC879780

Ht_TA3        KC879781

Ht_TA4        KC879782

Ht_TA5        KC879783

Ht_TA6        KC879784

Ht_TA7        KC879785

Ht_TA8        KC879786

Ht_TA9        KC879787

Ht_TA10       KC879788

Ht_MY1        KC879789                 1

Ht_MY2        KC879790                 5  12

Ht_MY3        KC879791                 1

Ht_MY4        KC879792

Ht_MY5        KC879793

Ht_MY6        KC879794                     1

Ht_MY7        KC879795                     1

Ht_MY8        KC879796                     1

Ht_MY9        KC879797                 1

Individuals/                           8  15

Mm_IS1        KC879787            1

Mm_IS2        KC879799

Mm_IS3        KC879800

Mm_IS4        KC879801

Mm_TA1        KC879802

Mm_TA2        KC879803

Mm_TA3        KC879804

Mm_MY1        KC879805            1

Mm_MY2        KC879806            1

Mm_MY3        KC879807            1

Mm_MY4        KC879808            1

Mra_MY5       KC879809            1

Mm_MY6        KC879810            1

Mm_MY7        KC879811            2

Mm_MD1        KC879812                         2          1

Mm_MD2        KC879813                         1

Mm_MD3        KC879814                         1

Mm_MD4        KC879815                         1

Mm_MD5        KC879816                         1          1

Mm_MD6        KC879817                                    1

Mm_MD7        KC879818                                    1

Mm_MD8        KC879819                                    1

Mm_MD9        KC879820                                    1

Individuals/                      9            6          6

Haplotype     Genebank   Individuals/
              Accession     haplotype

A1_IS1        KC879738              7

A1_IS2        KC879739              1

A1_IS3        KC879740              1

A1_IS4        KC879741              1

A1_IS5        KC879742             20

A1_IS6        KC879743              1

A1_IS7        KC879744              1

A1_MD1        KC879745              3

A1_MD2        KC879746              1

A1_MD3        KC879747              1

A1_MD4        KC879748              2

A1_MD5        KC879749              1

A1_MD6        KC879750              1

A1_MD7        KC879751              1

A1_MD8        KC879752              2

A1_MD9        KCK79753              2

A1_MD10       KC8 79754             1

A1_MD11       KC879755              1

A1_MD12       KC879756              1

A1_MD13       KC879757              1

A1_TA1        KC879758              1

A1_TA2        KC879759              1

A1_TA3        KC879760              1

A1_TA4        KC879761              1

A1_TA5        KC879762              2

A1_TA6        KC879763              1

A1_TA7        KC879764              1


Ht_TK1        KC879765              1

Ht_TK2        KC879766              1

Ht_TK3        KC879767              2

Ht_TK4        KC879768              1

Ht_TK5        KC879769              5

Ht_TK6        KC879770              1

Ht_TK7        KC879771              1

Ht_TK8        KC879772              1

Ht_TK9        KC879773              1

Ht_TK10       KC879774              2

Ht_TK11       KC879775              2

Ht_TK12       KC879776              2

Ht_TK13       KC879777              1

Ht_TK14       KC879778              1

Ht_TA1        KC879779              1

Ht_TA2        KC879780              1

Ht_TA3        KC879781              1

Ht_TA4        KC879782              1

Ht_TA5        KC879783              1

Ht_TA6        KC879784              1

Ht_TA7        KC879785              1

Ht_TA8        KC879786              1

Ht_TA9        KC879787

Ht_TA10       KC879788              1

Ht_MY1        KC879789              1

Ht_MY2        KC879790             21

Ht_MY3        KC879791              I

Ht_MY4        KC879792              1

Ht_MY5        KC879793              1

Ht_MY6        KC879794              1

Ht_MY7        KC879795              1

Ht_MY8        KC879796              1

Ht_MY9        KC879797              1


Mm_IS1        KC879787              1

Mm_IS2        KC879799              1

Mm_IS3        KC879800              1

Mm_IS4        KC879801              1

Mm_TA1        KC879802              I

Mm_TA2        KC879803              1

Mm_TA3        KC879804              1

Mm_MY1        KC879805              1

Mm_MY2        KC879806              1

Mm_MY3        KC879807              1

Mm_MY4        KC879808              1

Mra_MY5       KC879809              1

Mm_MY6        KC879810              2

Mm_MY7        KC879811              5

Mm_MD1        KC879812              3

Mm_MD2        KC879813              1

Mm_MD3        KC879814              I

Mm_MD4        KC879815              1

Mm_MD5        KC879816              2

Mm_MD6        KC879817              1

Mm_MD7        KC879818              1

Mm_MD8        KC879819              1

Mm_MD9        KC879820              1


Individual sampling sites listed in Table 1 and geographical
coordinates are available from the corresponding author
upon request.

Population genetic analyses

Antecaridina lauensis. Estimates of h (0.775) and [pi] (0.065) were mostly consistent across populations of A. lauensis, with the sole exception being Yoshino Cave (YOSH) on Ishigaki (Fig. 2A, Table 1). While no significant genetic structure was detected via pairwise [[PHI].sub.ST] or [S.sub.nn] analyses between A. lauensis populations in the southern Ryukyus and Minami-daito, the YOSH population was significantly different from all others in the Ryukyus (Table 3). Parsimony (TCS) analysis of the COI sequences produced two discrete networks, with individuals from the YOSH population occurring in both (Fig. 2C). One network consisted of 23 haplotypes and 48 individuals from the islands of Ishigaki, Tarama, and Minami-daito (Fig. 2B), whereas the other included four haplotypes from 10 individuals specific to the YOSH population on Ishigaki (Fig. 2C). Uncorrected p-distances among COI haplotypes were 0.16%-1.4% within a network and 12.7%-13.5% between the two networks. Tajima's D and Fu's F values were found to be significant and negative for sequences in the two networks (Table 2), suggesting recent population expansions in both groups. When COI sequences from A. lauensis and Antecaridina sp. sampled in Hawai'i, Christmas Island in the Indian Ocean (both supplied by Dr. Timothy J. Page, Griffith University, Queensland, Australia: unpubl. data), and East Timor (GenBank accessions EF173843--EF173847) from Craft et al. (2008) are included in the parsimony analysis, those from Hawai'i (n = 1) and Christmas Island (n = 2) were identical to the inferred ancestral haplotype found in the Ishigaki, Tarama, and Minami-daito populations (Fig. 2B), while a third network, composed of five haplotypes from the eight East Timor individuals (i.e., an Antecaridina sp.), was also recovered (data not shown).

Table 3.

Paitwise [[PHI].sub.ST] (below diagonal) and Snn (above
diagonal) estimates as measures of genetic differentiation
between populations of Antecaridina lauensis, Halocaridinides
trigonophthalma, and Metabetaeus minutus from the Ryukyu
Archipelago, Japan, based on cytochrome c oxidase subunit
1 (COI) sequences

             Population Site Code

            Antecaridina Site lauensis

           JR     SHIN     SHUG     YOSH

JK          -    0.454    0.525  0.747 *
SHIN    0.000        -    0.408  0.707 *
SHUG    0.000    0.000        -  0.727 *
YOSH  0.575 *  0.597 *  0.597 *        -

            Halocaridinides trigonophthalma

          TAG       IR     PUSH      MIN     YOSH
TAG         -    0.541  1.000 *  1.000 *    0.468
IR      0.000        -  1.000 *  1.000 *    0.552
PUSH  0.982 *  0.986 *        -    0.364  1.000 *
MIN   0.908 *  0.929 *  0.070 *        -  1.000 *
YOSH    0.000    0.000  0.982 *  0.901 *        -

               Metabetaeus minutus

           JR      MIY      KIK      FUT     YOSH

JR          -    0.389  1.000 *  1.000 *  1.000 *
MIY     0.028        -  1.000 *  1.000 *  1.000 *
KIK   0.956 *  0.951 *        -    0.392    0.244
FUT   0.947 *  0.940 *    0.000        -    0.325
YOSH  0.951 *  0.945 *    0.000    0.000        -

Site code: See Table 1.

* P < 0.05.

Halocaridinides trigonophthalma. For H. trigonophthalma, estimates of h spanned from 0.371 to 0.983, and with the exception of the one site on Taketomi, [pi] values were consistent among populations (Table 1). Significant genetic structure was found between those populations on the islands of Taketomi and Tarama relative to ones on the islands of Miyako, Irabu, and Ishigaki (Table 3), and three discrete networks were recovered from the parsimony analysis of H. trigonophthalma (Fig. 2D). The first corresponded to 9 haplotypes and 29 individuals from populations on Miyako, Irabu, and Ishigaki; the second was composed of 24 haplotypes and 30 individuals from the Taketomi and Tarama populations (Fig. 3A); and the third consisted of two individuals and one haplotype exclusive to the island of Taketomi (Fig. 2D). Here, p-distances among haplotypes in the same network ranged from 0.16% to 1.2%, while those between the three networks were from 9.52% to 19.2%. Two of the three H. trigonophthalma groups displayed evidence of recent population expansions as evident from significantly negative Tajima's D and Fu's [F.sub.s] values (Table 2).

Metabetaeus minutus. Estimates of haplotype diversity (h) for M. minutus ranged from 0.93 to 1.0, with nucleotide diversity ([pi]) being consistent among populations (Table 1). Pairwise [[PHI].sub.ST] or [S.sub.nn] values approaching, or at, their upper limit of 1.0 (Table 4) were estimated between the Southern Ryukyus and Minami-daito, implying strong genetic structure between populations separated by more than 600 km (Fig. 2A, B). On the other hand, no genetic structure was identified among M. minutus populations of the southern Ryukyus spanning a distance of about 200 km (Table 3). Two discrete networks (Fig. 2E) were recovered from the parsimony analysis: one represented M. minutus from the Southern Ryukyus, consisting of 14 haplotypes from 21 individuals collected on Ishigaki. Miyako, and Tarama islands; the second was from populations on Minami-daito, encompassing 9 haplotypes recovered from 12 individuals (Table 3). Uncorrected (p) distances among haplotypes within each network ranged from 0.16% to 1.4%, while those for haplotypes between the two networks ranged from 9.7% to 10.6%. Both Tajima's D and Fu's F values were significantly negative for M. minutus of the Southern Ryukyus, while those for populations on Minamidaito were not significant (Table 2).

Phylogenetic analyses

Analyses of A. lauensis, H. trigonophthalma, and M. minutus from the Ryukyus at the population level identified each as being composed of two to three distinct and divergent genetic lineages. To test whether each "species" was monophyletic as well as to infer the evolutionary relationships among lineages, 16S-rDNA sequences were generated for one to two individuals per lineage, as well as from specimens of the same or closely related species from outside the Ryukyus (Fig. 3A, Table 4), for phylogenetic analyses (Table 4). Maximum likelihood (ML) trees, inferred under the GTR model of evolution (as chosen by the AIC), for both datasets were well resolved with moderate-to-strong bootstrap support among branches (Figs. 3B, C).

Table 4. Additional caridean specimens and sequences
included in 16S-rDNA phylogenetic analyses

Taxonomic Group      Location          Accession  Source

Antecaridina sp.     East Timor        EF173754   Craft et al.,

Antecaridina         Christmas Island  EU123850   Page el al.,
lauensis                                          2008

                     Hawai'i [dagger]  -          Unpublished

                     Ishigaki          KC879824   This study

                     Minami-daito      KC879825   This study

Caridina rubella     Miyako            KC879826   This study

Halocaridina rubra   Hawai'i           EF173734   Craft el al.,

                     Hawai'i           EF173734   Craft el al.,

Halocaridinides      Miyako            KC879823   This study

                     Takclumi          KC879821   This study

                     Taketomi          KC879822   This study

Metabetaeus minutus  Christmas Island  KC879829   This study

                     Ishigaki          KC879828   This study

                     Minami-daito      KC879827   This study

Metabetaeus          Moorea            KC879831   This study
mcphersonae          [dagger][dagger]

Metabetaeus lohena   Hawai'i           KC879830   This study

Metabetaeus sp.      New Caledonia     FJ943435   Bracken et
                                                  al., 2009

Betaeus harrimani                      FJ943434   Bracken el al.

Macrobrachium        Okinawa           DQ194935   Liu. M., el
japonicum                                         al., 2007

[dagger] Sequence provided by Timothy Page, Griffith
University, Queensland Australia.

[dagger][dagger] Samples provided by Arthur Anker, Florida
Museum of Natural History.

In the analysis of the atyid dataset, both Antecaridina and Halocaridinides were monophyletic at the level of genera with strong (i.e., 98%-100% bootstrap) support (Fig. 3B). For Antecaridina, the lineage found on Ishigaki and Tarama in the southern Ryukyus as well as on Minami-daito was sister with moderate (i.e., 79% bootstrap) support to A. lauensis from Christmas Island in the Indian Ocean and Hawai'i in the central Pacific Ocean; the sequence divergence in 16S-rDNA was less than 1% across this geographic range (Fig. 3A). The second lineage, which was confined to the YOSH population on Ishigaki, was sister with strong (i.e., 93% bootstrap) support to an Antecaridina sp. from East Timor, which lies between the South China Sea and Indian Ocean (Fig. 3A). Notably, both groups of A. lauensis in the Ryukyus were more closely related to lineages from outside the archipelago then to one another. For the three genetic lineages of H. trigonophthalma, the two lineages restricted to Tarama and Taketomi formed a monophyletic group sister to the third lineage, which was found on Ishigaki, Miyako, and Irabu (Fig. 3B), with strong (i.e., 95%-100% bootstrap) support. Unfortunately, no specimens or genetic data for H. trigonophthalma from outside the Ryukyus could be obtained for this study.

Similar patterns were recovered from the phylogenetic analysis of the alpheid dataset. Specifically, Metabetaeus was monophyletic with strong (i.e., 100% bootstrap) support (Fig. 3C). Within Metabetaeus, all four M. minutus lineages were also monophyletic with relatively strong (i.e., 86% bootstrap) support to the exclusion of M. lohena (Hawai'i) and M. incphersonae (Moorea). For the two M. minutus lineages of the Ryukyus, the one from islands in the southern Ryukyus (i.e., Ishigaki, Miyako, and Tarama) was sister with strong (i.e., 99% bootstrap) support to M. minutus from Christmas Island in the Indian Ocean (Fig. 3A, C). The second lineage, apparently confined to the island of Minami-daito more than 600 km east of the southern Ryukyus, was sister with relatively strong (i.e., 87% bootstrap) support to a Metabetaeus sp. from New Caledonia in the southwest Pacific Ocean (Fig. 3A, B). As with the case for A. lauensis, both groups of M. minutus in the Ryukyus were more closely related to lineages from outside the archipelago then to one another. Furthermore, previously recognized morphological variation and character states also support the phylogenetic relationships inferred here. In this context, Anker (2010) noted differences in rostrum length and the angle of orbital teeth between M. minutus of Minami-daito and Christmas Island. In this study, specimens from the Minami-daito lineage conformed to the description of materials from the same locality, while those from the southern Ryukyus (i.e., Ishigaki, Miyako, and Tarama) lineage resemble the description of the Christmas Island specimens (Fig. Al, see Appenix) presented in Anker (2010).


Here, we applied a comparative phylogeographic approach to three caridean shrimp species toward differentiating the potential origins of anchialine crustacean biodiversity in the Ryukyu Archipelago. In this context, Antecaridina lauensis, Halocaridinides trigonophthalina, and Metabetaeus minutus were identified as comprising two to three lineages with considerable population structure and genetic divergence. If diversification within these three species followed the center of origin hypothesis and occurred within the Ryukyus, one would expect the evolutionary history of the A. lauensis, M. minutus, and H. trigonoph-Mainly lineages to be correlated with the sequential separation of the island groups in the archipelago as sea levels fluctuated during the Pleistocene. While phylogenetic patterns supporting the center of origin hypothesis of Indo-West Pacific fauna have been reported for a number of marine species in this geographic area (reviewed in Benzie, 1998; Carpenter et al., 2011), this does not appear to be the case for the anchialine species examined here. Instead, the phylogenetic relationships of the multiple lineages within each of these three anchialine carideans, coupled with regional oceanographic current patterns and geographic distributions, suggest the Ryukyus has been a sink to divergent lineages coming from independent source populations, consistent with the center of accumulation hypothesis.

Evidence for multiple colonizations of the Ryukyus by anchialine carideans

Whether the occurrence of related taxa across an island archipelago reflects diversification in situ or multiple colonizations can potentially be reconciled through phylogenetic reconstruction. For example, if island taxa are found to be monophyletic, a single colonization is favored and the speciose Drosophila (Grimaldi et al., 1990) and silverswords (Baldwin et al., 1991) of the Hawaiian Islands are exemplars of such a situation. On the other hand, paraphyly typically implies that multiple colonizations have occurred (Emerson, 2002). For A. lauensis and M. minutus in the Ryukyus, the two lineages within each species were not recovered as sister taxa in our phylogenetic analyses (Fig. 3). Rather, they exhibit paraphyletic relationships to A. lauensis and M. minutus lineages from outside (i.e., Christmas Island, East Timor, or New Caledonia) the Ryukyu Archipelago. Given these patterns, the three lineages of H. trigonophthalma found in the Ryukyus are likely the result of multiple colonizations from sources outside the archipelago as well. Similar phylogenetic patterns attributed to multiple colonizations have also been elucidated for a number of terrestrial (Harbaugh and Baldwin, 2007; Swenson et al., 2007; Nattier et al., 2011) and marine (Holland et al., 2004; Burridge et al., 2006) island taxa throughout the Pacific, including the Ryukyus (see Introduction).

Could the diversification of A. lauensis, M. minutus, and possibly H. trigonophthalma into multiple genetic lineages have occurred within the Ryukyus, with subsequent dispersal and colonization to regions outside the archipelago? Although phylogenetic patterns similar to those reported here would be recovered in such a scenario, this seems unlikely given the general oceanographic patterns of the region. Specifically, the Indo-West Pacific is dominated by the historically stable Kuroshio Current, which diverges from the North Equatorial Current east of the Philippine Islands, flowing northward into the Okinawa Trough (past the Ryukyus) and eventually out into the Pacific Ocean through the Tokara Strait (Ujiie et al., 2003). As the world's second-largest oceanic current (Shen et al., 2011), it is thought to have played a major role in generating the species diversity of the Indo-West Pacific seen today (e.g., Kojima et al., 1997; Mukai et al., 2009; Soeparno et al., 2012). For example, the North Equatorial and Kuroshio Currents contribute measurably to the dispersal of marine fishes from the central and southern Indo-West Pacific to Japan and the Ryukyus (e.g., Tsukamoto, 2006; Mukai et al., 2009). Similarly, the Kuroshio Current (along with the South China and North China Coastal Currents) apparently influenced the re-colonization of the Ryukyus by the flathead mullet. Magil cephalus, from southern refugia after Plio-Pleistocene sea-level changes (Shen et al., 2011). Given these recurring patterns, one could hypothesize that these strong northward currents aided in the dispersal and multiple colonizations of the Ryukyus by caridean species and lineages originating from anchialine habitats elsewhere in the Pacific Basin, most likely in the southern Indo-West Pacific.

As mentioned in the Introduction, climatic oscillations during the Pleistocene globally drove large fluctuations in sea-level stands, dramatically impacting the diversification and distributions of many species throughout the Indo-West Pacific (e.g., Barber et al., 2006; De Bruyn and Mather, 2007; Crandall et al., 2008; Fitzpatrick et al., 2011). For anchialine carideans, these cyclical fluctuations in sea levels likely influenced the availability of island, habitat, or both, as multiple "waves" of colonizers encountered the Ryukyu Archipelago. Assuming that higher and larger islands have been above sea level for longer time periods than low-lying and smaller ones, a strong correlation is apparent between current distributions of the anchialine caridean lineages examined here and island elevation and area in the Ryukyus. For example and consistent with this idea, the islands of Ishigaki, Miyako, and Irabu, with elevations of more than 100 m and areas greater than 200 [km.sup.2], appear to have been colonized by one of the H. trigonophthalma lineages at a time when sea levels were higher than present and smaller islands would have been submerged. As sea levels receded to present day conditions (or lower), a second (and third) H. trigonophthaltna lineage swept through the Ryukyus, colonizing the previously submerged islands of Taketomi and Tarama, both of which have much lower (i.e., <30 m) elevations and smaller (i.e., <15 [km.sup.2]) areas than Ishigaki, Miyako, or Irabu. The hypothesis of a recent colonization for these lower and smaller islands is supported by the fact that the H. trigonophthalma lineage found on both Taketomi and Tarama possesses the signal of a strong population expansion, as evident by the largest negative Tajima's D and Fu's [F.sub.s] values encountered in the study (Table 3), indicative of a potentially recent founder event. Furthermore, the only anchialine caridean lineage that exhibits no evidence of a recent population expansion (Table 3) is M. minutus found in the Daito Islands (i.e., Minami-daito and Kita-daito), which are thought to have migrated to their current location via plate tectonics and be considerably older (i.e., ~50 MYA; Kawana and Ohde, 1993; Ohde, 2007) than the islands in the Southern Ryukyus (i.e., ~10 MYA; see Introduction). Given this as well as the evolutionary relationship between the Minami-daito lineage and the M. minutus found on New Caledonia (Fig. 3), the anchialine habitats of Minami-daito may have been colonized by this alpheid when the islands were volcanically formed in the southern Indo-West Pacific near present-day New Guinea (Kawana and Ohde, 1993; Ohde, 2007) and prior to the emergence of (and habitat availability on) the southern islands in the Ryukyus. A similar pattern can be seen for A. lauensis, where one lineage is restricted to the island of Ishigaki and may represent an older colonization event, while the second lineage has an exceptionally impressive range of about 11,000 km, with an identical haplotype being shared among populations on Christmas Island (Indian Ocean), three islands in the Ryukyus (e.g., Ishigaki, Tarama, and Minami-daito), and Hawaii (Central Pacific), suggesting either a recent colonization or ongoing dispersal and gene flow across the Indian and Pacific Basins.

Population structure of anchialine carideans in the Ryukyus Archipelago

Despite having similar ecologies and broadly sympatric distributions within the Ryukyus, each of the A. lauensis, H. trigonophthalma, and M. minutus lineages exhibits radically different levels and scales of population structure and geographic range. One potential driver for this phylogeographical discordance among lineages and populations is subtle differences in intrinsic life-history characteristics. Such differences, particularly in life-history traits (i.e., egg size and larval stages), larval feeding mode (i.e., lecithotrophy vs. planktotrophy), and larval habitat have been implicated in influencing the dispersal potential and genetic structure of a number of caridean species from anchialine as well as stream habitats (Shokita, 1979; Page and Hughes. 2007; Craft et al., 2008; Russ et al., 2010). The significance of this is discussed in the following paragraphs.

For atyids such as A. lauensis and H. trigonophthalma, egg size is thought to be an effective and significant predictor of both dispersal ability and level of genetic structure (Shokita, 1979; Page and Hughes, 2007; Craft et al., 2008). For example, Caridina spp. from Australian streams possessing more restricted distributions and exhibiting higher levels of genetic structure typically have large (-1.6 mm) eggs, whereas those with extensive geographic ranges and low levels of genetic structure generally have relatively small (-0.4 mm) eggs (Page and Hughes, 2007). However, Halocaridinides, which produces relatively small eggs (-0.35 mm) with planktotrophic development (Fujita. unpublished), exhibits surprisingly strong genetic structure (i.e., approaching, or at, the maximum limit of 1.0) between islands (i.e., Ishigaki vs. Taketomi) separated by less than 5 km of shallow ocean. This is similar to 'short rostrum' Caridina rubella, another atyid possessing life-history traits conducive to dispersal (i.e., small egg size and planktotrophic larvae), from anchialine habitats on the island of Miyako (Weese et al., 2012), suggesting that the correlation between egg size and genetic structure in the Atyidae may not be as generally applicable as previously thought. As for A. lauensis, the species has historically been reported to have an "extremely disjunctive" distribution due to high dispersal abilities (Smith and Williams, 1981). The two A. lauensis lineages sampled to date represent opposite ends of the spectrum of range distributions, with one being confined to a single anchialine habitat (i.e., YOSH) of a single island (i.e., Ishigaki) and the other inhabiting multiple anchialine habitats in both the Indian and Pacific Oceans. Unfortunately, no information is currently available on egg size, larval feeding mode, or larval habitat for A. lauensis. However, it can be hypothesized that the lineage with a highly restricted range differs significantly in these traits from the other lineage that has a much broader distribution, and laboratory reproductive studies of these two A. lauensis lineages would directly address this possibility. In any case, one vexing question is this: How did multiple A. lauensis and H. trigonophthalma lineages successfully colonize the Ryukyus on several occasions from likely distant sources when current populations remain in isolation over small spatial scales (i.e., an apparent absence of short-distance dispersal)? Local retention of larvae within nearshore waters might help explain this level and pattern of genetic structure (Weese et al., 2012). It remains to be determined, however, to what extent additional biotic (i.e., differences in other reproductive or ecological/physiological traits (e.g., salinity tolerance, dietary differences, resource partitioning, competition) or abiotic (i.e., local oceanic currents, geography, and/or geology) factors contribute to this situation as well.

Although little is known about the life history of the alpheid M. ininutus, field and laboratory observations suggest that the species produces many (i.e., 70-170) small (0.72-0.52 mm) eggs that develop as planktotrophic larvae (Fujita, unpublished). In contrast to the lecithotrophic (i.e., yolk-bearing) larvae produced by atyids like Halocaridina rubra (Craft et al., 2008), planktotrophic larvae are considered to be less energy constrained because of the ability to feed during planktonic dispersal, typically leading to low genetic structure between populations (reviewed by Palumbi, 1994). Given this, it is not surprising that populations of the M. minutus lineage from the Southern Ryukyus are homogenized across the islands of Miyako, Tarama, and Ishigaki, which span a distance of about 200 km (Fig. 2). The lack of population structure at this scale is similar to that in a few other anchialine species like the neritiliid snail Neritilia cavernicola (Kano and Kase. 2004) in the Philippines and Metabetaeus lohena (Russ et al., 2010) in the Hawaiian Islands, where both species also possess planktotrophic larvae and exhibit little-to-no genetic differentiation over ranges of about 200 km. Additionally, the two populations of M. minutus sampled from the lineage confined to Minarni-daito exhibited no genetic structure across the island, which is again similar to M. lohena populations occurring on any single island in Hawai'i (Russ et al., 2010). However, the fact that no M. minutus haplotypes are shared between populations in the Southern Ryukyus and Minarni-daito implies isolation by distance over greater (i.e., >600 km) geographic scales. Collectively, this suggests that while some caridean species such as Metabetaeus spp. (i.e., minutus or lohena) may be "good" dispersers in "ecological" timescales when habitats or islands are in close (i.e., ~200 km) proximity, dispersal and potential colonization for the three anchialine shrimp examined here becomes more of an "evolutionary sweepstake" event as habitats or islands become further apart (i.e., from <5 km to >600 km), with subsequent isolation following successful colonization.

Distribution and cryptic diversity of anchialine shrimp of the Indo-West Pacific

The anchialine fauna of the Indo-West Pacific is dominated by caridean species, with 11 being recorded throughout the Pacific Basin (de Grave and Sakihara, 2011). While some, such as H. rubra. M. lohena, and Procaris hawaiana Holthuis, 1973 (Decapoda: Procarididae), are characterized as endemic to single archipelagos (e.g., the Hawaiian Islands), many others, such as A. lauensis, H. trigonoph-thalma, M. minutus, Calliasmata pholidota Holthuis, 1973 (Decapoda: Hippolytidae), Parhippolyte uveae Borradaile, 1899 (Decapoda: Barbouriidae), and Periclimenes pholeter Holthuis, 1973 (Decapoda: Palaemonidae), are thought to have extremely widespread and disjunct distributions throughout the Indo-West Pacific (reviewed by Maciolek, 1983). However, the emerging patterns of multiple genetic lineages with strong population structure (Santos, 2006; Craft et al., 2008; Santos and Weese, 2011; Weese et al., 2012; this study) imply that the caridean biodiversity of anchialine habitats in the Pacific Basin may be vastly underestimated, with many previously described species actually representing cryptic species complexes. For example, both A. lauensis and M. minutus in this study appear to represent complexes composed of at least (given the current sampling) two potentially divergent species in the Ryukyus alone. Furthermore, the population structure and genetic divergence found within H. trigonophthalma of the Ryukyus suggest a complex of at least three species. Likewise, cryptic species complexes have previously been described for the anchialine atyids Caridina rubella (two potential species) on Miyako in the Ryukyus (Weese et al., 2012) and Halocaridina rubra (eight potential species) in the Hawaiian Islands (Santos 2006; Craft et al., 2008). In fact, of the six Pacific anchialine caridean species studied to date from a population genetic perspective, 18 distinct and divergent lineages have been identified, with only M. lohena failing to be a cryptic species complex (Russ et al., 2010). Given this, it would not be surprising if genetic analyses of other widely distributed Pacific anchialine crustaceans, such as C. pholidota, P. pholeter, and P. uveae, reveal cryptic species complexes as well. Although additional morphological studies will be required to resolve the taxonomic status of the lineages within each complex, the restricted distributions of these potential "species" should be taken into consideration when developing conservation and management strategies for anchialine carideans and their habitats throughout the Indo-West Pacific.


This work was funded in part by a National Science Foundation (NSF) East Asia and Pacific Summer Institutes Fellowship OISE-0913667 (DAW) and NSF DEB-0949855 (SRS). We thank Dr. Timothy J. Page (Griffith University, Australia) and one anonymous reviewer for providing valuable comments that improved the manuscript. This manuscript represents contributions #107 and #14 to the Auburn University (AU) Marine Biology Program and Molette Biology Laboratory for Environmental and Climate Change Studies, respectively.

Literature Cited

Akey, J. M., M. A. Eberle, M. J. Rieder, C. S. Carlson, M. D. Shriver, D. A. Nicerson, and L. Kruglyak. 2004. Population history and natural selection shape patterns of genetic variation in 132 genes. PLoS ONE 2: e286.

Anker, A. 2010. Metabetaeus Borradaile, 1899 revisited, with description of a new marine species from French Polynesia (Crustacea: Decapoda: Alpheidae). Zooraxa 2552: 37-54.

Baldwin, G. B., D. W. Kyhos, J. Dvorak, and G. D. Carr. 1991. Chloroplast DNA evidence for a North American origin of the Hawaiian silversword alliance (Asteraceae). Proc. Natl. Acad. Sci. USA 88: 1840-1843.

Banner, A. H., and D. M. Banner. 1960. Contributions to the knowledge of shrimp of the Pacific Ocean. Part VII. On Metahetaeus Borradaile, species from Hawaii. Pac. Sci 14: 299-303.

Barber, P. H., M. V. Erdmann, and S. R. Palunibi. 2006. Comparative phylogeography of three codistributed stromatopods: origins and timing of regional lineage diversification in the coral triangle. Evolution 60: 1825-1839.

Benzie, J. A. H. 1998. Genetic structure of marine organisms and SE Asian biogeography. Pp. 197-209 in Biogeography and Geological Evolution of SE Asia, R. Hall and J. D. Holloway, eds. Backhuys. Leiden, The Netherlands.

Bird, C. E., B. S. Holland, B. W. Bowen, and R. J. Toonen. 2007. Contrasting phylogeography in three endemic Hawaiian limpets (Cellana spp.) with similar life histories. Mol. Ecol. 16: 3173-3187.

Bird, C. E., B. S. Holland. B. W. Bowen, and R. J. Toonen. 2011. Diversification in broadcast-spawning sympatric Hawaiian limpets (Celiana spp.). Mal. Ecol. 20: 2128-2141.

Borradaile, L. A. 1899. On the Stromaopoda and Macura brought by Dr. Willey from the South Seas. Pp. 395-428 in Zoological Results Based on Material from New Britain, New Guinea, Loyalty Islands and Elsewhere. Collected During the Years 1895, 1896 and 1897, A. Willey, ed. Cambridge University Press. Cambridge.

Bracken. H. D., A. Toon, D. L. Felder, J. W. Martin, NI. Finley, J. Rasmussen, F. Palero, and K. A. Crandall. 2009. The decapod tree of life: compiling the data and moving toward a consensus of decapod evolution. Arthropod Syst. Phylogeny 67: 99-116.

Briggs, J. C. 2000. Centrifugal speciation and centers of origin. J. Biogeogr. 27: 1183-1188.

Buhay, J. E. 2009. "COI-Like" sequences are becoming problematic in molecular systematic and DNA barcoding studies. J. Crustacean Biol. 29:96-110.

Burridge, C. P., R. Melendez, and B. S. Dyer. 2006. Multiple origins of the Juan Fernandez Kelpfish fauna and evidence for frequent and unidirectional dispersal of Cirrhitoid fishes across the South Pacific. Syst. Biol. 55: 566-578.

Cabezas, P., F. Alda. E. Macpherson, and A. Machordom. 2012. Genetic characterization of the endangered and endemic squat lobster Munipodsis polymotpha from Lanzarote (Canary Islands): management implications. ICES J. Mar. Sci. doi:10.1093/icesjms/fss062.

Cai, Y. and S. Shokita. 2006. Atyid shrimps (Crustacea: Decapoda: Caridea) of the Ryukyu Islands, southern Japan, with descriptions of two new species. J. Nat. Hist. 40: 2123-2172.

Carpenter, K. E., P. H. Barber, E. D. Crandall, M. C. A. Ablan-Lagman, Ambariyanto, G. N. Mahardika, B. M. Manjaji-Matsumoto, M. A. Juinio-Menez, M. D. Santos, C. J. Starger, and A. H. A. Toha. 2011. Comparative phylogeography of the Coral Triangle and implications for marine management. J. Mar. Biol. doi: 10.1155/2011/396982.

Clement, M., D. Posada, and K. A. Crandall. 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9: 1657-1659.

Cook, B. D, A. M. Baker, T. J. Page, S. C. Grant, J. H. Fawcett, D. A. Harwood, and J. M. Hughes. 2006. Biogeographic history of an Australian freshwater shrimp. Paratya australiensis (Atyidae): the role life history transition in phylogeographic diversification. Mol. Ecol. 15: 1083-1093.

Craft, J. D., A. D. Russ, M. N. Yamamoto, T. Y. Iwai, S. Haw J. Kahiapo, C. T. Chong, S. Ziegler-Chong, C. Muir, Y. Fujita, D. A. Polhemus, R. A. Kinzie, and S. R. Santos. 2008. Islands under islands: the phylogeography and evolution of Halocaridina rubra Holthuis, 1963 (Crustacean: Decapoda: Atyidae) in the Hawaiian archipelago. Limnol. Oceanogr. 53: 675-689.

Crandall, E. D., M. A. Frey, R. K. Grosberg, and P. H. Barber. 2008. Contrasting demographic history and phylogeographical patterns in two Indo-Pacific gastropods. Mol. Ecol. 17: 611-626.

Crandall, K. A., A. R. Templeton, and C. F. Sing. 1994. Intraspecific phylogenetics: problems and solutions. Pp. 273-297 in Models in Phylogeny Reconstruction, R. W. Scotland, D. J. Siebert, and D. M. Williams, eds. Clarendon Press, Oxford.

De Bruyn, M., and P. B. Mather. 2007. Molecular signatures of Pleistocene sea-level changes that affected connectivity among freshwater shrimp in Indo-Australian waters. Mol. Ecol. 16: 4295-4307.

de Grave, S., and T. S. Sakihara. 2011. Further records of the anchia-line shrimp. Periclintenes pholeter Holthuis, 1973 (Crustacea, Decapoda, Palemonidac). Zootaxa 2903: 64-68.

De Haan, W. 1833-1850. Crustacean. Pp.1-243 in Fauna japonica sive descripto anirnalium, quae in itinere per japoniam, jussu et aus piciis superiorum, qui summum in India Batava Imperium Tenent, Suscepto, Annis 1823-1830 Collegit, Norts, Obervationibus et Adum-brationibus Illustravit. P.F. von Siebold. ed. Lugduni-Batavorum, Leiden, Netherlands.

Edmondson, C. H. 1935. New and rare Polynesian Crustacea. Bernice P. Bishop Mus. Occas. Papers 10: 3-40.

Emerson, B. C. 2002. Evolution on oceanic islands: molecular phylogenetic approaches to understanding pattern and process. Mol. Ecol. 11: 951-966.

Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin (version 3.0): An integrated software package for population genetics data analysis. Evol. Bioinform. Online 1: 47-50.

Fitzpatrick, J. M., D. B. Carlon, C. Lippe, and D. R. Robertson. 2011. The West Pacific diversity hotspot as a source or a sink for new species'? Population genetic insights from the Indo-Pacific parrot fish Scams rubroviolaceus. Mol. Ecol. 20: 219 -234.

Folmer, O., M. Black, W. Hoch, R. Lutz, and R. Vrijenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit 1 from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294-299.

Fu, Y. 1997. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147: 915-925.

Fujino, T., and S. Shokita. 1975. Report on some new atyid shrimps (Crustacea. Decapoda, Caridea) from the Ryukyu Islands. Bull. Sci. Engr. Unit., Ryukyus Math. Nat. Sci. 18: 93-113.

Fujita, Y. 2007. Decapod crustaceans inhabiting springs on Miyako Islands, the Ryukyus. Japan. Bulletin of the Miyakojima City Museum 11: 89-110.

Fajita, Y., and H. Sunagawa. 2008. Cavernicolous and terrestrial decapod crustaceans of Tarama-jima Island, the Ryukyu Islands, southwestern Japan. Bulletin of the Miyakojima City Museum 12: 53-80.

Grimaldi, D. A., L. H. Throckmorton, and T. Okada. 1990. A phylogenetic, revised classification of genera in the Drosophilidae (Diptera). Bull. Ain. Mus. Nat. Hist, 197: 1-139.

Guindon, S., and 0. Gascuel. 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52: 696-704.

Harbaugh. D. T., and B. G. Baldwin. 2007. Phylogeny and biogeography of the Sandalwoods (Santalurn, Santalaceae): repeated dispersals throughout the Pacific. Am. J. Bot. 94: 1028-1040.

Hikida. T., and J. Motokawa. 1999. Phylogeographic relationships of the skinks of the genus Eumeces (Repitilia: Scincidae) in East Asia. Pp. 231-247 in Tropical Island Herpetofauna--Origin, Current Diversity, and Conservation. H. Ota ed. Elsevier, Amsterdam.

Holland, B. S., M. N. Dawson. G. L. Crow, and D. K. Hofmann. 2004. Global phylogeography of Cassiopea (Scyphozoa: Rhizostomeae): molecular evidence for cryptic species and multiple invasions of the Hawaiian Islands. Mar. Biol. 145: 1119-1128.

Holthuis, L. B. 1973. Caridean shrimps found in land-locked saltwater pools at four Indo-West Pacific localities (Sinai Peninsula. Funafuti Atoll, Maui and Hawai'i Islands) with the description of one new genus and four new species. Zool. Meded. (Leiden) 128: 1-48.

Hudson, R. R. 2000. A new statistic for detecting genetic differentiation. Genetics 155: 2011-2014.

Hunter, R. L., M. S. Webb, T. M. Biffe, and R. A. Bremer. 2008. Phylogeny and historical biogeography of the cave-adapted shrimp genus Typhlatya (Atyidae) in the Caribbean Sea and western Atlantic. J. Biogeogr. 35: 67-75.

Ivey, J. L., and S. R. Santos. 2007. The complete mitochondrial genome of the Hawaiian anchialine shrimp Halocaridina rubra Holthuis, 1963 (Crustacea: Decapoda: Atyidae). Gene 394: 35-44.

Kano, Y., and T. Kase. 2004. Genetic exchange between anchialine cave population by means of larval dispersal: the case of a new gastropod species Neritilia cavernicola. Zool. Scr. 33: 423-437.

Kawana, T., and S. Ohde. 1993. A short reconnaissance of Okino-Daito-Jima Island in the Northern Philippine Sea: implication for Quaternary crustal movements of the raised almost-table reef. Bulletin of the College of Education. Univ. Ryukyus 43: 57-69 [in Japanese with English abstract].

Kimura, M. 2000. Paleogeography of the Ryukyu Islands. Tropics 10: 5-24.

Koba, M. 1980. Distribution and age of the marine terraces and their deposits in the reef capped Ryukyu Islands, Japan. Quat. Res. 8: 189-208.

Kojbna, S., R. Segawa, and I. Hayashi. 1997. Genetic differentiation among populations of the Japanese turban shell Turbo (Batillus) cornutus corresponding to warm currents. Mar. Ecol. Prog. Ser. 150: 149-155.

Kornai, T., and Y. Fujita. 2005. A new stygiobiont species of Macro-brachium (Crustacea: Decapoda: Caridea: Palaemonidae) from an anchialine cave on Miyako Island, Ryukyu Islands. Zootaxa 1021: 13-27.

Konishi, K., and K. Sudo. 1972. From Ryukyus to Taiwan. Kagaku 42: 221-230.

Lee, C. S., G. C. Bisbee, L. D. Lu, and R. S. Hilde. 1980. Okinawa trough: origin of a back-arc basin. Mar. Geol. 35: 219-241.

Liggins, L., D. G. Chapple, C. H. Daugherty, and P. A. Ritchie. 2008. Origin and postcolonization evolution of the Chatham Islands skink (Oligosoma nigriplantarel. Mol. Ecol. 17: 3290-3305.

Lin, S. M., C. A. Chen, and K. Y. Lue. 2002. Molecular phylogeny and biogeography of the grass lizards genus Takydromus (Reptilia: Lacerticlae) of east Asia. Mol. Phylogenet. Evol. 22: 276-288.

Liu, M. Y., Y. X. Cal, and C. S. Tzeng. 2007. Molecular systematics of the freshwater prawn genus Macrobrachium Bate. 1868 (Crustacea: Decapoda: Palaemonidae) inferred from mt DNA sequences, with emphasis of East Asian Species. Zool. Stud. 46: 272-289.

Liu, X., F. Hayashi, and D. Yang. 2008. Systematics and biogeography of the fishily genus Parachatdiodes (Megaloptera: Corydalidae) endemic to the east Asian Islands. Syst. Entomol. 33: 560-578.

Lopez, J. V., N. Yuhki, R. Masuda, W. Modi, and S. J. O'Brien. 1994. Nunn, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat. J. Mol. Eva 39: 174-190.

Maciolek, J. A. 1983. Distribution and biology of Indo-Pacific insular hypogeal shrimp. Bull. Mar. Sci. 33: 606-618.

Maciolek. J. A. 1986. Environmental features and biota of anchialine pools on Cape Kinau, Maui. Hawaii. Stygologia 2: 119-129.

Maekawa, K., N. Lo, 0. Kitade, T. Miura, and T. Matsumoto. 1999. Molecular phylogeny and geographic distribution of wood-feeding cockroaches in East Asian islands. Mol. Phylogenet. Evol. 13: 360376.

Meijaard, E. 2003. Mammals of south-east Asian islands and their Late Pleistocene environments. J. Biogeogr. 30: 1245-1257.

Mukai, T., S. Kakamura, and M. Nishida. 2009. Genetic population structure of a reef goby. Bathygobius cocosensis, in the northwest Pacific. Ichthyol. Res. 56: 380-387.

Muraji, M., N. Arakaki, S. Ohno, and Y. Hirai. 2008. Genetic variation of the green chafer. Anomala albopilosa (Hope) (Coleoptera: Scarabaeidea) in the Ryukyu Island of Japan detected by mitochondrial DNA sequences. Appl. Entomol. Zool. 43: 299-306.

Murphy, N. P. and C. M. Austin. 2005. Phylogenetic relationships of the globally distributed firewater prawn genus Macrobrachium (Crustacea: Decapoda: Palaemonidae): biogeography, taxonomy and the convergent evolution of abbreviated larval development. Zool. Scr. 34: 187-197.

Myers, N., R. A. Mittermeier, C. G. Mittermeier, G. A. B. Da Fonseca, and J. Kent. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.

MyIroie, J. E., and J. R. Mylroie. 2011. Void development on carbonate coasts: creation of anchialine habitats. Hydrobiologia 1: 15-32.

Naruse, T., and H. Tamura. 2006. A first record of anchialine crab of the genus Orcovita Ng and Tomascik. 1994 (Decapoda: Brachyura: Varunidae) from Japan, with description of the species. Limnology 7: 147-151.

Nattier, R., T. Robillard, L. Desutter-Grandcolas, A. Couloux, and P. Grandcolas. 2011. Older than New Caledonia emergence? A molecular phylogenetic study of the eneopterine crickets (Orthoptera: Grylloidea). J. Biogeogr. 38: 2195-2209.

Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York.

Ohde, S. 2007. Estimating the ocean environmental change based on coral/coral reef chemistry. Transactions of the Research Institute of Oceanochemistry 20: 55-76 (in Japanese).

Page, T. J., and J. M. Hughes. 2007. Radically different scales of phylogeographic structuring within cryptic species of freshwater shrimp (Atyidae: Caridina). Limnol. Oceanogr. 52: 1055-1066.

Page, T. J., K. Rintelen, and J. M. Hughes. 2007. An island in the stream: Australia's place in the cosmopolitan world of Indo-West Pacific freshwater shrimp (Decapoda: Atyidae: Caridinu). Mol. Phylogenet. Evol. 43: 645-659.

Page, T. J., W. F. Humphreys, and J. M. Hughes. 2008. Shrimps down under: evolutionary relationships of subterranean crustaceans from Western Australia (Decapoda: Atyidae: Stygiocaris). PLoS ONE 3: el618.

Palumbi, S. R. 1994. Genetic divergence, reproductive isolation, and marine speciation. Annu. Rev. Ecol. Syst. 25: 547-572.

Posada, D. 2006. Modeltest Server: a web-based tool for the statistical selection of models of nucleotide substitution online. Nucleic Acids Res. 34: W700-W703.

Posada, D., and K. A. Crandall. 1998. Modeltest: testing the model of DNA substitution. Bioninformatics 14: 817-818.

Rambaut, A. 2008. Se-Al. [Online]. Available [2013, 15 August].

Rambaut, A. 2009. FigTree [Online]. Available [2013, 15 August].

Rathbun, M. J. 1904. Decapod Crustaceans of the Northwest Coast of North America. Harriman Alaska Expedition, Doubleday, New York.

Rozas, J., J. Sanchez-DelBarrio. X. Messeguer, and R. Rozas. 2003. DnaSP. DNA polymoprphism analyses by the coalescent and other methods. Bioinformatics 19: 2496-2497.

Russ, A. R., S. R. Santos, and C. Muir. 2010. Genetic population structure of an anchialine shrimp. Metabetaeus lohena (Crustacea: Alpheidae), in the Hawaiian Islands. Rev. Biol. Trop. 58: 159-170.

Santos, S. R. 2006. Patterns of genetic connectivity among anchialine habitats: a case study of the endemic Hawaiian shrimp Halocaridina rubra on the Island of Hawaii. Mol. Ecol. 15: 2699-2718.

Santos. S. R., and D. A. Weese. 2011. Rocks and clocks: linking geologic history and rates of genetic differentiation in anchialine organisms. Hydrobiologia 677: 54-63.

Shen, K., B. W. Jamandre, C. Hsu, W. Tzeng, and J. Durand. 2011. Plio-Pleistocene sea level and temperature fluctuation in the northwestern Pacific promoted speciation in the globally-distributed flathead mullet Mugil cephalus. BMC Evol. Biol. 11: 83.

Shokita, S. 1979. The distribution and speciation of the island water shrimps and prawns from the Ryukyu Islands. Bulletin of the College of Science, Univ. Ryukyus 28: 193-278.

Smith, M. J., and W. D. Williams. 1981. The occurrence of Antecaridina lauensis (Edmondson) (Crustacea, Decapoda, Atyidae) in the Solomon Islands. Hydrobiologia 85: 49-58.

Soeparno, Y. Nakamura, T. Shibuno, and K. Yamaoka. 2012. Relationship between pelagic larval duration and abundance of tropical fish on temperate coasts of Japan. J. Fish Biol. 80: 346-357.

Suzuki, H. 2001. A comment on the geological formation of the Mishima Islands (Takeshima, loujima and Kuroshima) as inferred from their freshwater crustacean faunas. Occasional Papers of Kagoshima University 34: 136-140.

Swenson, U., J. Munzinger, and I. V. Barash. 2007. Molecular phylogeny of Planchonella (Sapotaceae) and eight new species from New Caledonia. Taxon 56: 329-354.

Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585-595.

Tamura, K., and M. Nei. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10: 512-526.

Templeton, A. R., K. A. Crandall, and C. F. Sing. 1992. A cladistics analysis of phenotypic association with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132: 767-782.

Thompson, J. D. 1997. The ClustA1_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-7882.

Tsukamoto, K. 2006. Oceanic biology: spawning of eels near a seamount. Nature 439: 929.

Ujiie, Y., H. Ujiie. A. Taira, T. Nakamura, and K. Oguri. 2003. Spatial and temporal variability of surface water in the Kuroshio source region. Pacific Ocean, over the past 21,000 years: evidence from planktonic foraminifera. Mar. Micropaleontol. 49: 355-364.

Vandergast, A. G., R. G. Gillespie, and G. K. Roderick. 2004. Influence of volcanic activity on the population genetic structure of Hawaiian Tetragnatha spiders: fragmentation, rapid population growth and the potential for accelerated evolution. Mol. Ecol. 13: 1729-1743.

Weese, D. A., Y. Fujita. M. Hidaka, and S. R. Santos. 2012. The long and short of it: genetic variation and population structure of the an-chialine atyid shrimp Caridina rubella on Miyako-jima, Japan. J. Crustacean Biol. 32: 53-64.

Whitelegge, T. 1897. The Crustacea. Memoirs of the Australian Museum 3: 127-151.

Received 22 May 2013: accepted 15 July 2013.

* To whom correspondence should be addressed at Museum of Natural History. University of Michigan. Ann Arbor MI 48109. E-mail:


(1) Department of Biological Sciences and Molette Biology Laboratory for Environmental and Climate Change Studies, Auburn University, 101 Life Sciences Building, Auburn, Alabama 36849; (2) University Education Center, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213. Japan; (3) Marine Learning Center, 2-95-101 Miyagi, Chatan-cho, Okinawa 904-0113, Japan; (4) Cell and Molecular Biosciences Peak Program., Auburn University, 101 Life Sciences Building, Auburn, Alabama 36849
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Author:Weese, David A.; Fujita, Yoshihisa; Santos, Scott R.
Publication:The Biological Bulletin
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Geographic Code:9JAPA
Date:Sep 1, 2013
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