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Cryptic species diversity in the genus Allactaga (Rodentia: Dipodidae) at the edge of its distribution range.

Abstract. Present study aimed to address molecular diversities of the small five-toed jerboa Allactaga elater Lichtenstein, 1825, and the Toussi jerboa Allactaga toussi Darvish et al., 2008, in marginal geographic distribution of the genus in Iran. The study involved 35 individuals of A. elater and A. toussi, from the east and central parts of Iranian plateau. The two probabilistic phylogenetic algorithms, Maximum Likelihood (ML) and Bayesian Inference (BI), applied on 68 sequences of the two mitochondrial genes (34 cytochrome b and 34 cytochrome c oxidase subunit 1), retrieved reciprocal monophyly of the two species. Independent species status of A. elater and A. toussi is further evident from their sympatry in eastern Iran. Each of these species was further subdivided into two deeply divergent phylogeographic lineages within Iranian plateau, showing high level of genetic divergence ranging from 7-10.7 % for cytb and 7.8-12.4 % for cox1 genes. Such values exceed the intraspecific level of variation in rodents.

Key words: Allactaga elater, Allactaga toussi, cox1, cytb, molecular phylogeny

Introduction

Five-toad jerboas are typical inhabitants of arid areas in Asia and north-eastern Africa. Twelve species were recognized in Wilson & Reeder (2005) and classified as a genus Allactaga. The diploid number of chromosomes is stable across the genus and molecular data are available only for few regions and species groups (Arslan et al. 2012, Dianat et al. 2013, Krystufek et al. 2013). Taxonomy has therefore been entirely based on morphology although it is known that characters used in species delimitation (e.g. size, colour, and shape of glance penis) are subjected to the intraspecific variations (Shenbrot 2009). Recent phylogenetic reconstructions, based on molecular markers, questioned the validity of established taxonomy at the level of species and of genera. Lebedev et al. (2013) proposed split into up to five genera and other analyses (Dianat et al. 2013, Krystufek et al. 2013) showed that number of species may be higher than that of conservative 12 species in Wilson & Reeder (2005).

We address in this paper the small five-toed jerboa A. elater (Lichtenstein, 1825) which was recently split in Iran into two species. The new species A. toussi Darvish et al., 2008 was recognized on morphological ground but its distinctness from A. elater received support in molecular reconstruction (Dianat et al. 2013). Molecular tools have the potential to facilitate both the identification of known species and the discovery of unrecognized cryptic diversity (Hebert et al. 2003, Jaarola et al. 2004). Herewith we provide evidence on deep divergence within A. elater and A. toussi, which may be indicative of further cryptic species in this group.

Material and Methods

Our study utilized 35 specimens of small five-toed jerboas from the eastern and central regions of Iran (Fig. 1 and Table 1). Specimens were classified either as A. elater or as A. toussi on the basis of morphological characteristics provided by Darvish et al. (2008); body mass, external (HBL, TL, EL and FL) and cranial measurements are larger in A. toussi than A. elater. Moreover, external characteristics are obviously discernable in these two species; inner surface of ear is dark in A. toussi, while it is light in A. elater, hind sole margin is naked in A. toussi, while it covers with black dense setae in A. elater (for more details see Darvish et al. 2008). Museum vouchers are deposited in the Zoological Museum of Ferdowsi University of Mashhad (ZMFUM), Iran. Whole genomic DNA of Allactaga was extracted from 96 % ethanol-preserved tissues using salt standard extraction method (Bruford et al. 1992). Polymerase Chain Reactions were performed using L7 and H6 primers for cytb gene (Montgelard et al. 2002), and VF1d and VR1d for cox1 gene (Ivanova et al. 2006). Purified PCR products were sequenced commercially by Macrogen Company, Republic of South Korea. 34 specimens were successfully sequenced. Sequences were edited manually using CodonCode aligner software (CodonCode Corporation) and aligned with Clustal W (Thompson et al. 1997) algorithm, using BioEdit 7.0.5 (Hall 1999). Genetic distances were analyzed assuming Kimura 2 parameter (K2P) model with 10000 bootstraps in Mega v6 (Tamura et al. 2013).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Phylogenetic analyses were performed on a combined multiple sequence alignment including 68 sequences obtained in this study (34 cox1, and 34 cytb), and 75 sequences (34 cox1, and 41 cytb) belonging to five species of Allactaga which were retrieved from GenBank (Dianat et al. 2013, Krystufek et al. 2013). The best fitting model of nucleotide substitution was estimated, using jModeltest 0.1.1 (Posada 2008). Bayesian Inference (BI), was performed under the General Time Reversible model (GTR) with a proportion of invariable sites (I) and a gamma distribution (G) using MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003). Four Monte Carlo Markov chains were run simultaneously for 4000000 generations. The trees were sampled every [100.sup.th] generation after removing the first 5000 trees as the burn-in stage. Branch support was assessed as Bayesian Posterior Probability (BPP). Maximum Likelihood analysis was performed based on the Akaike Information Criterion (AIC), with the assumed model. The branch support of the ML tree was assessed as bootstrap value (BP) with 200 replicates. We considered a BPP [greater than or equal to] 0.95 as "good" and BPP = 0.90-0.95 as "moderate" supports, while BP > 90 % as "good" support, and BP = 80-90 % as "moderate" support, in line with other authors (e.g. Krystufek et al. 2009). The phylogenetic trees were rooted with red squirrel, Sciurus vulgaris (AJ238588) as an out-group (Reyes et al. 2000).

Results

Together with data reported in Dianat et al. (2013), 40 cytb and 34 cox1 haplotypes are known for lesser five-toed jerboas. Of the 911 bp-long cytb sequence, 218 polymorphic sites (23.92 %) were found, 182 (83.48 %) of which were parsimony informative. Of the 620 bp-long cox1 sequence, 241 (38.87 %) and 183 (75.93 %) sites were polymorphic and parsimony informative sites, respectively. No stop codons, insertions or deletions were observed in the alignments.

We constructed BI and ML trees for the two mitochondrial genes independently (cytb, cox1) and for the concatenated sequence. Because of congruence between the outputs only the BI tree drawn for combined sequences is shown (Fig. 2). Seven highly supported (BPP = 1.00) lineages are eminent. Allactaga hotsoni was basal in the tree and williamsi + euphratica were in a sister position against the small five-toed jerboas. All specimens of small five-toed jerboas were in two lineages which matched their morphological classification. In northeastern Iran ranges of these two species overlapped and in Bojnord, Torbat Jam, and Sarakhs, they were sympatric. Each of the two small five-toed jerboas? species was further structured into two sub-lineages. Sub-lineages were strictly allopatric in A. toussi, being confined to north-eastern Iran (A. toussi the east) and to the area between the Zagros and Elbruz Mountain ranges (A. toussi the west). The two sub-lineages of A. elater were allopatric in the extreme north-eastern Iran, but were found in sympatry in Golestan. K2P genetic distances between the species of five-toed jerboas ranged between 12.6 and 20.7 % for cytb and between 13.4 and 20.9 % for cox1. Corresponding values for the sub-lineages of A. elater and A. toussi were 10.7 and 7.0 % for cytb, and 12.4 and 7.8 % for cox1 (Table 2).

Discussion

Our results confirmed association between the morphotype and the molecular profile in small five-toed jerboas classified as A. elater and A. toussi. Cytb divergence between them (12.6 %) was above the values reported for intraspecific divergences in rodents (Baker & Bradley 2006). Also importantly, these two jerboas were broadly sympatric in a large area of north-eastern Iran. It is therefore beyond doubt that elater and toussi are two distinct species.

Of no lesser interest are divergences within both, A. toussi and A. elater. In both species, the intraspecific cytb distances (7.0 and 10.7 % for toussi and elater, respectively) exceeded the intraspecific divergences in rodents ([less than or equal to] 4.7 %; Baker & Bradley 2006), being well within the range of K2P distances found between species. Since the two lineages of A. elater were sampled from the same place in Golestan, one may assume for them to be at least parapatric. Although we believe that cryptic species are most likely involved in both cases, we refrain at this stage from taxonomic conclusions. First of all, nuclear genetic markers have to be included into analyses, sampling in the zone of overlap has to be intensified, and morphological variation of glans penis is to be taken into consideration for these highly divergent sublineages. Following recent reports on cryptic species richness in five-toed jerboas (Paralactaga; Krystufek et al. 2013), our results further emphasize the extent of undetected species richness in dipodids. It seems that the phenomenon is widespread, if not overwhelming and that cryptic species will follow to be reported along with progress of molecular screening in the family. Small five-toed jerboas seem a particularly good candidate, considering large range, high number of subspecies and categorical divergence evidenced by the morphology of glans penis. Noteworthy, significant portion of variation takes place outside Iran since only one of the two groups of subspecies discerned from glans penis was reported for the country (Shenbrot et al. 2008a).

Allactaga vinogradovi as the closest relative to A. elater (Lebedev et al. 2013) has not been sequenced so far. Information on nucleotide sequences in A. vinogradovi is essential to get an unbiased insight into the phylogenetic structuring of the entire group of small five-toed jerboas (tentatively classified as Microalactaga; Lebedev et al. 2013) and particularly the relationships between A. vinogradovi and A. toussi. It came as a surprise that A. toussi is more widespread in Iran than is A. elater.

Acknowledgements

We would like to thank Prof. Boris Krystufek for his valuable comments, and two anonymous reviewers whose comments and suggestions greatly improved the paper. This study was funded by grant number 1.19727 for studying fauna of North Khorasan Province to JD.

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Samira MOSHTAGHI (1), Jamshid DARVISH (1,2), Omid MIRSHAMSI (1,3) and Ahmad MAHMOUDI (1*)

(1) Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran; e-mail: a.mahmoudi.bio@gmail.com

(2) Rodentology Research Department, Institute of Applied Zoology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

(3) Zoological Innovations Research Department, Institute of Applied Zoology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

Received 2 January 2016; Accepted 29 April 2016

(*) Corresponding Author
Table 1. Details of the sample localities for Allactaga were included
in phylogenetic analysis. Museum vouchers are deposited in Zoological
Museum of Ferdowsi University of Mashhad (ZMFUM).

Species            Locality      Museum voucher No.   Cytb accession no.

A. elater 1        Golestan              2749             KX219811
                                         2674             JQ954928
                     Gonabad             3524             KX219808
                     Gonbad              1905             KX219809
                     Bojnord             2842             KX219812
                                         2862             KX219813
                                         2897             KX219815
                     Kashmar             1412             KX219805
                                         1377             KX219804
                                         1374             KX219814
                                         2084             KX219806
                                         2128             JQ954931
                                         1429             JQ954932
                                         2704             KX219810
                    Torbate
                    Heydarie             1740             KX219807
 A. elater 2        Golestan             2738             KX219817
                                         2751             KX219818
                                         2732             KX219816
                                         2675             JQ954927
                                         2676             JQ954929
                                         2677             JQ954930
                     Sarakhs             3547             KX219819
                                         3548             KX219820
                                         3554             KX219821
                   Torbate Jam           3601             KX219822
                                         3602             KX219823
                                         3603             KX219824
                                         3604             KX219825
                                         3609             KX219826
                                         3613             KX219827
A. toussi West       Tehran              2745                    -
                                         2680             KX219836
                                         2679             JQ954935
                                         2680             JQ954936
                     Esfahan             2678             JQ954934
                     Hamedan             4503             KX219837
A. toussi East       Sarakhs             3558             KX219830
                                         3553             KX219831
                                         3557             KX219829
                   Torbate Jam           3614             KX219835
                     Mashhad            T1045             AJ389534
                                         2694             JQ954954
                                         2695             JQ954955
                                         2696             JQ954956
                                         1415             JQ954957
                                         1416             JQ954938
                                         1418             JQ954958
                     Sarakhs             2130             JQ954959
                                         1431             JQ954933
                    Sabzevar             1438             KX219834
                      Tabas              1434             KX219833
                     Bojnord             2864             KX219828
                                         2875             KX219832

Species         Cox1 accession no.            Reference

A. elater 1        KX219845                   Present study
                    JQ954893                 Dianat et al. 2013
                    KX219842                   Present study
                    KX219843                   Present study
                    KX219846                   Present study
                    KX219847                   Present study
                    KX219849                   Present study
                    KX219839                   Present study
                    KX219838                   Present study
                    KX219848                   Present study
                    KX219840                   Present study
                    JQ954902                 Dianat et al. 2013
                    JQ954900                 Dianat et al. 2013
                    KX219844                   Present study
                    KX219841                   Present study
 A. elater 2        KX219851                   Present study
                    KX219852                   Present study
                    KX219850                   Present study
                    JQ954894                 Dianat et al. 2013
                    JQ954895                 Dianat et al. 2013
                    JQ954896                 Dianat et al. 2013
                    KX219853                   Present study
                    KX219854                   Present study
                    KX219855                   Present study
                    KX219856                   Present study
                    KX219857                   Present study
                    KX219858                   Present study
                    KX219859                   Present study
                    KX219860                   Present study
                    KX219861                   Present study
A. toussi West      KX219871                   Present study
                    KX219870                   Present study
                    JQ954898                 Dianat et al. 2013
                    JQ954899                 Dianat et al. 2013
                    JQ954897                 Dianat et al. 2013
                        -                      Present study
A. toussi East      KX219864                   Present study
                    KX219865                   Present study
                    KX219863                   Present study
                    KX219869                   Present study
                        -                    Dianat et al. 2013
                    JQ954918                 Dianat et al. 2013
                    JQ954919                 Dianat et al. 2013
                    JQ954920                 Dianat et al. 2013
                    JQ954921                 Dianat et al. 2013
                        -                    Dianat et al. 2013
                    JQ954922                 Dianat et al. 2013
                    JQ954923                 Dianat et al. 2013
                    JQ954901                 Dianat et al. 2013
                    KX219868                   Present study
                    KX219867                   Present study
                    KX219862                   Present study
                    KX219866                   Present study

Table 2. The K2P (mean [+ or -] SD) estimates of intraspecific and
interspecific cytb and cox1 divergences in five species of Allactaga.

Description of node                        Cytb: K2P ([+ or -] SD)

A. elater /A. toussi                       12.6 [+ or -] 0.9
A. elater /A. hotsoni                      15.9 [+ or -] 1.2
A. elater /A. euphratica                   16.2 [+ or -] 1.0
A. elater /A. williamsi                    15.4 [+ or -] 1.1
A. elater /A. sibirica                     18.6 [+ or -] 1.4
A. toussi /A. hotsoni                      16.5 [+ or -] 1.2
A. toussi /A. euphratica                   16.4 [+ or -] 1.1
A. toussi /A. williamsi                    15.5 [+ or -] 1.3
A. toussi /A. sibirica                     17.3 [+ or -] 1.3
A. hotsoni /A. euphratica                  18.3 [+ or -] 1.2
A. hotsoni /A. williamsi                   17.9 [+ or -] 1.4
A. hotsoni /A. sibirica                    18.4 [+ or -] 1.5
A. euphratica /A. williamsi                15.0 [+ or -] 1.0
A. euphratica /A. sibirica                 20.7 [+ or -] 1.4
A. williamsi /A. sibirica                  18.7 [+ or -] 1.5
A. toussi: toussi the east/toussi the west  7.0 [+ or -] 0.8
A. elater: elater 1/elater 2               10.7 [+ or -] 1.1

Description of node                          Cox1: K2P ([+ or -] SD)

A. elater /A. toussi                         13.5 [+ or -] 1.3
A. elater /A. hotsoni                        18.1 [+ or -] 1.4
A. elater /A. euphratica                     20.2 [+ or -] 1.8
A. elater /A. williamsi                      18.4 [+ or -] 1.6
A. elater /A. sibirica                       18.9 [+ or -] 1.7
A. toussi /A. hotsoni                        13.4 [+ or -] 1.2
A. toussi /A. euphratica                     18.5 [+ or -] 1.8
A. toussi /A. williamsi                      17.1 [+ or -] 1.5
A. toussi /A. sibirica                       19.1 [+ or -] 1.9
A. hotsoni /A. euphratica                    20.9 [+ or -] 1.8
A. hotsoni /A. williamsi                     18.3 [+ or -] 1.5
A. hotsoni /A. sibirica                      19.9 [+ or -] 1.7
A. euphratica /A. williamsi                  14.6 [+ or -] 1.6
A. euphratica /A. sibirica                   19.6 [+ or -] 1.9
A. williamsi /A. sibirica                    19.5 [+ or -] 1.9
A. toussi: toussi the east/toussi the west    7.8 [+ or -] 1.1
A. elater: elater 1/elater 2                 12.4 [+ or -] 1.4
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Author:Moshtaghi, Samira; Darvish, Jamshid; Mirshamsi, Omid; Mahmoudi, Ahmad
Publication:Folia Zoologica
Article Type:Report
Geographic Code:7IRAN
Date:Jul 1, 2016
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