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, Kryštufek 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, Kryštufek 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).

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).

Fig. 1.

Map showing collection sites of the specimens used in this study. Different symbols refer to phylogenetic sub-lineages of A. elater, and A. toussi. (The approximate distribution of small five-toed jerboa (grey shadow) follows IUCN database, Shenbrot et al. 2008b;  http://dx.doi.org/10.2305/IUCN.UK.2008.RLTS. T853A13083932.en).

Fig. 2.

Phylogenetic relationships among five species of Allactaga obtained from concatenated dataset (cytb and cox1). Bayesian Inference and Maximum Likelihood analyses pose an identical topology. The first and second numbers on the branches correspond to posterior probability (BPP) and bootstrap (BP) values in the BI and ML tree analyses, respectively. (^*Sequences were downloaded from GenBank; published by Dianat et al. 2013). The symbols correspond to those in Figure 1.

Table 2.

The K2P (mean ± SD) estimates of intraspecific and interspecific cytb and cox1 divergences in five species of Allactaga.

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, Kryštufek 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^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 ≥ 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. Kryštufek 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 north-eastern 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 (≤ 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 sub-lineages. Following recent reports on cryptic species richness in five-toed jerboas (Paralactaga; Kryštufek 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.