Cryptic species diversity in the genus Allactaga (Rodentia: Dipodidae) at the edge of its distribution range.
Key words: Allactaga elater, Allactaga toussi, cox1, cytb, molecular phylogeny
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).
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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).
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).
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.
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: firstname.lastname@example.org
(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|
|Date:||Jul 1, 2016|
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