- Research article
- Open Access
Multilocus phylogeny and cryptic diversity in Asian shrew-like moles (Uropsilus, Talpidae): implications for taxonomy and conservation
© Wan et al.; licensee BioMed Central Ltd. 2013
Received: 1 June 2013
Accepted: 15 October 2013
Published: 25 October 2013
The genus Uropsilus comprises a group of terrestrial, montane mammals endemic to the Hengduan and adjacent mountains. These animals are the most primitive living talpids. The taxonomy has been primarily based on cursory morphological comparisons and the evolutionary affinities are little known. To provide insight into the systematics of this group, we estimated the first multi-locus phylogeny and conducted species delimitation, including taxon sampling throughout their distribution range.
We obtained two mitochondrial genes (~1, 985 bp) and eight nuclear genes (~4, 345 bp) from 56 specimens. Ten distinct evolutionary lineages were recovered from the three recognized species, eight of which were recognized as species/putative species. Five of these putative species were found to be masquerading as the gracile shrew mole. The divergence time estimation results indicated that climate change since the last Miocene and the uplift of the Himalayas may have resulted in the diversification and speciation of Uropsilus.
The cryptic diversity found in this study indicated that the number of species is strongly underestimated under the current taxonomy. Two synonyms of gracilis (atronates and nivatus) should be given full species status, and the taxonomic status of another three potential species should be evaluated using extensive taxon sampling, comprehensive morphological, and morphometric approaches. Consequently, the conservation status of Uropsilus spp. should also be re-evaluated, as most of the species/potential species have very limited distribution.
Major Disagreements in the taxonomy of Uropsilinae
Allen, 1938 
Ellerman & Morrison-Scott, 1951 
Hoffmann, 1984 
Nowak, 1999 
Cranbrook, 1960 
Hutterer, 2005 
Corbet, 1980 
Hoffmann & Lunde, 2008 
Honacki et al. 1982 
(andersoni a )
The mountains of southwest China and adjacent areas harbor an extremely high biodiversity . Two nonexclusive hypotheses, including a pre-Pleistocene diversification model and a Pleistocene refugia model have been proposed to explain the expansion of endemic species . Under the pre-Pleistocene diversification model, the uplifting of the Qinghai-Tibetan Plateau (QTP) in association with climate changes boosted allopatric speciation (e.g., [19–21]). Conversely, Quaternary glacial-interglacial cycles caused repeated shifts in distribution ranges , which may also have motivated diversification and speciation in isolated mountain chains [23–25]. Given that Uropsilinae might have evolved for approx. 30 million years , this genus could be a promising model to test these two hypotheses.
In this study, we sampled Asian shrew-like moles throughout their distribution range, sequenced both mitochondrial and nuclear loci, and adopted molecular phylogenetic and species delimitation approaches to represent a systematic framework of the genus Uropsilus. Our goals are the following: (i) to examine the proposed taxonomy based on morphology/morphometrics; (ii) to assess the evolutionary relationships among living taxa; and (iii) to test alternative scenarios of species/diversification patterns within the genus.
We obtained 6,330 bp sequences for each voucher specimen, including 1,985 bp mitochondrial (CYT B [1,140 bp] and 12S rRNA [845 bp]) and 4,345 bp nuclear (ADORA3 [321 bp], ATP7A [675 bp], BDNF [555 bp], BMI1 [313 bp], CREM [389 bp], PLCB4 [331 bp], RAG1 [1,010 bp], and RAG2 [751 bp]) sequences. A total of 552 sequences were deposited in GenBank with Accession nos. from KF777818 to KF778377 (Additional file 1: Table S1). No premature stop codon was observed in the coding regions of the protein coding genes examined. The mitochondrial genes showed relatively higher genetic polymorphisms than the nuclear genes (Additional file 2: Table S2). Note that the BMI1 gene had no variable site and was thus not used in the species delimitation or network tree reconstruction.
Gene trees and divergence times
Species delimitation and species trees
Mitochondrial vs. nuclear genes
Although incongruences between mitochondrial and nuclear or between different nuclear genes have been observed in many studies (e.g., ), the mitochondrial gene trees and PBS analyses suggested that in our case mitochondrial genes still represent good genetic markers. When both mitochondrial and nuclear genes alone failed to fully resolve the relationships, combined mitochondrial and nuclear genes using both concatenated and coalescent approaches recovered identical topologies and were robustly supported. It follows that a combination of both rapidly evolving mitochondrial and conservative nuclear genes appears to be the best approach to resolve ancient but closely spaced divergences.
Species delimitation and taxonomic reappraisal
The accuracy of molecular species delimitation has been discussed several times [34, 35]. BPP has been recognized as the most accurate approach when compared to other coalescent-based methods like ABC and SpeDeSTEM . Recently, Miralles and Vences found that the number of putative species strikingly ranged from 9 to 34, depending on the method implemented . Although BPP was not recognized as the best approach in their study, the results could be affected by small sample size of each putative species and the small number of loci (see  for detail). Despite the uncertainty of the number of putative species recognized, molecular data represent only a crude estimate rather than a final conclusion and should be diagnosed using comprehensive morphological characters.
Splits and BPP recognized 10 and 8 putative species, respectively. It is of note that clades A + B and G + H were recognized as one species each in the BPP analyses, even the pairwise K2P distances of CYT B were higher than 0.100. This value is higher than the distance between sister species U. aequodonenia and U. andersoni (0.085) and the average genetic distance between sister species of mammals (0.081; ). This result may be due to the small sample size of clade A (n = 2) and potential gene flow (see ) because A and B are sympatrically distributed and G and H were not supported as monophyletic by nuclear genes (Figure 1b). On the other hand, U. sp. 2, U. sp. 3 were recognized as putative species even though the K2P distance between them and their sister taxa were very low (K2P = 0.013-0.039), which might due to mitochondrial introgression or different lineage sorting scenarios. We treated U. spp. 2 and 3 as putative species here, as the BPP analyses suggested there was no recent gene flow. Regardless of the ambiguity of species delimitation, the high pairwise genetic distances and the polyphyly of previously known U. gracilis strongly suggested that number of species of Uropsilus has been underestimated.
With respect to the previous taxonomy of Uropsilus, dental formulas have been used as a key for species diagnosis. Four dental formulas have been observed, and U. gracilis and U. investigator share the same dentition (Additional file 3: Figure S1; ). Our results revealed that six polyphyletic taxa (species/putative species) share the same dental formula (i2/1,c1/1,p4/4,m3/3 = 38; Additional file 3: Figure S1). Within the six species/putative species, clade E was sampled from the type locality of U. gracilis, and can be safely assigned to this species (Figure 4). The type locality of U. investigator is distributed to the west of the Mekong River, geographically close to the clades A and B, thus we assigned clade A + B to U. investigator. Clades G + H and C consist of populations from the type localities of atronates and nivatus, respectively, and these two taxa should most likely be recognized as valid species. There is no available name for spp. 1–3, and their taxonomic status should be evaluated based on extensive sampling and comprehensive morphological/morphometric comparisons.
Divergence pattern of Uropsilus
The results of our divergence time estimation suggested that neither the pre-Pleistocene speciation nor the Pleistocene “speciation pump” hypothesis could exclusively explain the speciation/diversification pattern. Indeed, the diversification of Uropsilus may have been affected by periodical orogenic processes and climate change. Prevailing trends toward cooling and desiccation in the late Miocene (i.e., Messinian salinity crisis; [27, 38]) and the Pliocene/Pleistocene boundary  have been well documented, and leading to the diversification of humid-dwelling taxa (e.g., [40, 41]). Therefore, global cooling may be responsible for the splits of the Asian shrew-like moles at 6.18 Ma and 2.40 Ma. Similarly, geological studies have supported rapid uplifts of QTP at 3.6 Ma, 1.8 and 1.1 Ma [28, 30], which may also have resulted in the diversifications of the genus.
The distribution of different species/putative species showed a strong geographic pattern, which could be partly due to the extremely complex topography, understory habitats, and low dispersal ability of the animals [42, 43]. Nonetheless, at least four species/putative species, including U. aequodonenia, U. andersoni, U. soricipes, and U. sp.1, are distributed in the western Sichuan mountains, indicating a very complex geographic history. Extensive sampling is required to uncover the vicariant, migration, and speciation history patterns.
Cryptic diversity and conservation implications
Identifying cryptic diversity is essential for the accurate assessment of genetic diversity and conservation planning [10, 44]. Cryptic divergence and strong geographic structures have been observed in other endemic taxa [45, 46], indicating that topography has strong effect on diversification, particularly with regard to small and sedentary animals. The current conservation statuses of Uropsilus spp. are all considered as “Least Concern” or “Data Deficient” (IUCN Red List Category; ), due to their presumed wide distribution and/or large population size . However, our results indicated that unrecognized species exist within Uropsilus, and most of the species/putative species have very limited distribution. Indeed, because of global warming and continuous habitat loss, the species diversity of the genus may be actually threatened. Therefore, re-evaluation of the endangered categories relying on a new systematic background is warranted; at present, these putative species should be considered as evolutionary significant units and taken into consideration for conservation planning.
In the present study, we obtained sequences of Uropsilus throughout their distribution in the mountains of southwest China. We reconstructed a robust phylogeny for this most primitive talpid genus and found cryptic diversity. Five putative species were determined in addition to the five recognized species. We suggested that atronates and nivatus should be recognized as full species, and a comprehensive morphological diagnosis is warranted for three unidentified species. Moreover, the conservation statues should re-evaluated, as most of the species/putative have limited distribution. Finally, the divergence of the genus may be affected by climatic changes and tectonic activities, providing clues for the expansion of endemic fauna.
Sample collection and ethics statement
Samples used in this study
Mt. Qinling, Shannxi
Mt. Emei, Sichuan
Mt. Jinfo, Chongqing
Mt. Gaoligong, Yunnan
Mt. Yulong, Yunnan
Mt. Diancang, Yunnan
Mt. Biluo, Yunnan
Mt. Yongde, Yunnan
Mt. Wuliang, Yunnan
Primers used for PCR and sequencing
Primer sequences (5′-3′)s
Annealing temperature (°C) *
Sequence assembling and alignment
Sequences were assembled and edited using DNASTAR Lasergene version 7.1. All genes were aligned in MUSCLE  and further examined by eye in MEGA5 . In addition, the CYT B sequences determined in a recent study representing Uropsilus aequodonenia, U. andersoni, U. gracilis and U. soricipes were downloaded from GenBank . Sequences of Talpa altaica of the subfamily Talpinae and Sorex araneus of the family Soricidae were chosen as outgroup taxa (Additional file 1: Table S1).
Phylogenetic analyses and divergence time estimation
To reconstruct the phylogenetic relationships of Uropsilus, ML analyses were performed using RAxML v7.3.2 , and Bayesian inference (BI) was performed using BEAST v1.7.5 . The phylogenetic analyses were conducted on the following four datasets: 1) a mitochondrial combined gene dataset; 2) a nuclear gene combined dataset; 3) an all gene combined dataset; 4) the same as dataset 3 but with the CYT B sequences downloaded from GenBank removed (Additional file 6). We used a partitioning strategy to incorporate the variation in evolutionary processes among different sites . The best-fit partitioning scheme and the appropriate model of DNA evolution for each partition were determined in PartitionFinder v1.0 . The alignment was partitioned by gene and by codon position. Twelve models of molecular evolution (K80, HKY, TrNef, TrN, SYM, GTR, K80 + G, HKY + G, TrNef + G, TrN + G, SYM + G, GTR + G) were compared and ranked by the Bayesian Information Criterion (BIC) . The best partitioning scheme and substitution models are given in Additional file 7. The ML analyses were performed using the CIPRES Science Gateway . We selected the GTR + gamma model for each partition and the rapid Bootstrapping algorithm (Stamatakis A, Hoover P, Rougemont J: A Rapid Bootstrap Algorithm for the RAxML, Web-Servers, unpublished) and ran 500 bootstrap replicates. The Bayesian phylogenetic trees were calculated in BEAST v1.7.5 . We employed relaxed uncorrelated exponential clock models that allowed the rate in each branch to evolve independently . The combined mitochondrial fragment and each nuclear gene were given specific exponential clock models, and a Continuous-time Markov chains (CTMCs) model was employed as a prior for each clock model . Each analysis used a random staring tree, a birth-death tree prior and the program’s default prior distribution of the model parameters. Each analysis was run for 50 million generations with a sampling interval of 5,000 was conducted. Trace v1.5  was used to confirm the effective sample sizes (ESSs) as greater than 200 and the first 30% of the generations were treated as burn-in. The BEAST analyses were repeated four times. To assess the support for the mitochondrial and nuclear genes at each node, partitioned branch support scores (PBSs; ) were calculated using TreeRot v2.0  and PAUP 4.0b10 . We performed this analysis using dataset 4. We further constructed network trees for each nuclear gene using NETWORK v4.611; the nuclear genes were unphased with DnaSP v5.10  using the algorithms provided by PHASE , and the unphased haplotypes were used to construct median-joining haplotype networks . We ran the MP calculation post-processing option to delete all of the superfluous median vectors and chose one of the shortest trees.
The molecular divergence time was estimated using BEAST v1.7.5. Because mitochondrial genes evolve much faster than nuclear genes , missed nuclear genes will lead to inaccurate estimates of branch lengths and divergence times. Therefore, we used dataset 4 for the molecular dating analyses with U. aequodonenia and U. andersoni excluded. For the fossil-calibrated age constraints, lognormal and exponential distributions were used to account for uncertainty in fossil calibrations . Three calibrations were used. (i) The split of the most recent common ancestor (MRCA) shared by Talpidae and Erinaceidae + Soricidae was at approximately 73 (78–68) million years ago (Ma) . We set up the prior using a lognormal distribution (offset = 63, mean = 10.4, standard deviation = 0.24), such that the mean age was at 73 Ma and the 95% upper boundary was at 78 Ma. (ii) The oldest known Talpini was from the early Oligocene (Palaeogene mammal unit MP 21)  at approximately 33.9-32.6 Ma ; thus, we set up the prior using an exponential distribution (offset = 32.6, mean = 10.8; 32.6*0.333) . (iii) We employed the oldest known U. soricipes from 2.4-2.0 million-year-old strata (early Pleistocene) in Hubei Province, China, and set up the prior using an exponential distribution, (offset = 2.0, mean = 0.67 2.0*0.333) [76, 77].
Species delimitation and species tree estimation
We conducted species delimitation analyses using the “splits” v1.0-14 package (SPecies LImits by Threshold Statistics) for the R statistical environment and the programe BPP v2.2. When using splits, the time-calibrated concatenated gene tree was used as the input tree, whereas the number of putative species was identified using a generalized mixed Yule-coalescent model (GMYC) . We further used BPP v2.2 to conduct a Bayesian species delimitation [79, 80]. This software provides the most accurate molecular species delimitation to date , and the mixing of the Markov chain is improved in the new version v2.2 . The computational simulation demonstrated that the correct species models could be inferred with high posterior probabilities when 5 or 10 sequences from each population were sampled . A previous study found that a mis-specified guide tree can result in strong support for more species . Therefore, we estimated the species tree using the *BEAST model implemented in BEAST (see below), and assigned the 56 specimens to 10 putative species based on the results of splits (see Results). The analyses were performed using both the mitochondrial-nuclear combined data (dataset 4) and the nuclear genes alone (dataset 2). Gamma prior G (6, 6,000) was used on the population size parameters (θs), and the age of the root in the species tree (τ0) was assigned gamma prior G (4, 1,000). Multiple runs were performed using both the species delimitation algorithm 0 and algorithm 1. “Locusrate = 1” specifying the random-rates model of Burgess and Yang , or “Heredity = 1” allowing θ to vary among loci, were also used but not at the same time. The analyses for each data set were repeated 12 times. Each rjMCMC was run for 1 million generations and sampled every 10 generations after discarding 10,000 generations as pre-burn-in.
The species trees were estimated using the *BEAST model  in BEAST v1.7.5. Because the *BEAST model uses very different assumptions from a concatenated gene tree estimation, we did not use the partitioning scheme derived from Partitionfinder. Instead, we gave 12S rRNA, each codon position of CYT B gene and each nuclear gene a different substitution model. The best-fit models were calculated using jModeltest  and are provided in Additional file 7. Because *BEAST requires that all species have at least one sequence at each locus, we used dataset 4 for the *BEAST analyses. The 56 specimens were assigned to 8 or 10 putative species based on the results of splits and BPP (see Results). We used the same priors as described above. Each analysis was run for 100 million generations and sampled every 10,000 generations.
DNA sequences: accession numbers are provided in Additional file 1: Table S1.
We appreciate the constructive comments and suggestions from the editor Dr. Miguel Vences and the anonymous reviewer. We thank Chi Zhang for discussing the BPP analyses in detail. We thank Nai-Qing Hu, Wei-Wei Zhou, Wan-Sheng Jiang, Feng Dong, Lan-Ping Zheng and Min-Sheng Peng for their meaningful suggestions on the laboratory work and earlier versions of this manuscript. This work was supported by grants from the National Natural Science Foundation of China (No. 31301869 and 31272276), Knowledge Innovation Program of the Chinese Academy of Sciences (KSZD-EW-2-011), and the Fund of State Key Laboratory of Genetics Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences (GREKF11-03).
This work is dedicated to Dr. Robert S. Hoffmann, who greatly contributed to the science of mammalogy in China.
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