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      Small Mammal Investigation in Spotted Fever Focus with DNA-Barcoding and Taxonomic Implications on Rodents Species from Hainan of China

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          Abstract

          Although mammals are a well-studied group of animals, making accurate field identification of small mammals is still complex because of morphological variation across developmental stages, color variation of pelages, and often damaged osteological and dental characteristics. In 2008, small mammals were collected for an epidemiological study of a spotted fever outbreak in Hainan, China. Ten species of small mammals were identified by morphological characters in the field, most using pelage color characters only. The study is extended here, in order to assess whether DNA barcoding would be suitable as an identification tool in these small mammals. Barcode clusters showed some incongruence with morphospecies, especially for some species of Rattus and Niviventer, so molecular delineation was carried out with an expanded dataset of combined cytochrome b (Cyt- b) and cytochrome c oxidase subunit I (COI) sequences. COI sequences were successfully amplified from 83% of collected mammals, but failed in all specimens of Suncus murinus, which were thus excluded in DNA barcoding analysis. Of note, ten molecular taxonomic units were found from samples of nine morphologically identified species. Accordingly, 11 species of small mammals were present in the investigated areas, including four Rattus species, three Niviventer species, Callosciurus erythraeus, Neohylomys hainanensis, Tupaia belangeri, and Suncus murinus. Based on the results of the phylogenetic and molecular delineation analyses, the systematic status of some rodent species should be redefined. R. rattus hainanicus and R. rattus sladeni are synonyms of R. andamanensis. R. losea from China and Southeast Asia comprises two independent species: R. losea and R. sakeratensis. Finally, the taxonomic status of three putative species of Niviventer should be further confirmed according to morphological, molecular and ecological characters.

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          Bayesian species delimitation using multilocus sequence data.

          In the absence of recent admixture between species, bipartitions of individuals in gene trees that are shared across loci can potentially be used to infer the presence of two or more species. This approach to species delimitation via molecular sequence data has been constrained by the fact that genealogies for individual loci are often poorly resolved and that ancestral lineage sorting, hybridization, and other population genetic processes can lead to discordant gene trees. Here we use a Bayesian modeling approach to generate the posterior probabilities of species assignments taking account of uncertainties due to unknown gene trees and the ancestral coalescent process. For tractability, we rely on a user-specified guide tree to avoid integrating over all possible species delimitations. The statistical performance of the method is examined using simulations, and the method is illustrated by analyzing sequence data from rotifers, fence lizards, and human populations.
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            SPECIATION IN MAMMALS AND THE GENETIC SPECIES CONCEPT.

            We define a genetic species as a group of genetically compatible interbreeding natural populations that is genetically isolated from other such groups. This focus on genetic isolation rather than reproductive isolation distinguishes the Genetic Species Concept from the Biological Species Concept. Recognition of species that are genetically isolated (but not reproductively isolated) results in an enhanced understanding of biodiversity and the nature of speciation as well as speciation-based issues and evolution of mammals. We review criteria and methods for recognizing species of mammals and explore a theoretical scenario, the Bateson-Dobzhansky-Muller (BDM) model, for understanding and predicting genetic diversity and speciation in mammals. If the BDM model is operating in mammals, then genetically defined phylogroups would be predicted to occur within species defined by morphology, and phylogroups experiencing stabilizing selection will evolve genetic isolation without concomitant morphological diversification. Such species will be undetectable using classical skin and skull morphology (Morphological Species Concept). Using cytochrome-b data from sister species of mammals recognized by classical morphological studies, we estimated the number of phylogroups that exist within mammalian species and hypothesize that there will be >2,000 currently unrecognized species of mammals. Such an underestimation significantly affects conclusions on the nature of speciation in mammals, barriers associated with evolution of genetic isolation, estimates of biodiversity, design of conservation initiatives, zoonoses, and so on. A paradigm shift relative to this and other speciation-based issues will be needed. Data that will be effective in detecting these "morphologically cryptic genetic species" are genetic, especially DNA-sequence data. Application of the Genetic Species Concept uses genetic data from mitochondrial and nuclear genomes to identify species and species boundaries, the extent to which the integrity of the gene pool is protected, nature of hybridization (if present), and introgression. Genetic data are unique in understanding species because the use of genetic data 1) can quantify genetic divergence from different aspects of the genome (mitochondrial and nuclear genes, protein coding genes, regulatory genes, mobile DNA, microsatellites, chromosomal rearrangements, heterochromatin, etc.); 2) can provide divergence values that increase with time, providing an estimate of time since divergence; 3) can provide a population genetics perspective; 4) is less subject to convergence and parallelism relative to other sets of characters; 5) can identify monophyly, sister taxa, and presence or absence of introgression; and 6) can accurately identify hybrid individuals (kinship and source of hybrid individuals, F(1)s, backcrosses, direction of hybridization, and in concert with other data identify which hybrids are sterile or fertile). The proposed definition of the Genetic Species Concept is more compatible with a description of biodiversity of mammals than is "reproductively isolated species." Genetic profiles of mammalian species will result in a genetic description of species and mammalian diversity, and such studies are being accelerated by technological advances that reduce cost and increase speed and efficiency of generating genetic data. We propose that this genetic revolution remain museum- and voucher specimen-based and that new names are based on a holotype (including associated tissues) deposited in an accredited museum.
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              Sequence-based species delimitation for the DNA taxonomy of undescribed insects.

              Cataloging the very large number of undescribed species of insects could be greatly accelerated by automated DNA based approaches, but procedures for large-scale species discovery from sequence data are currently lacking. Here, we use mitochondrial DNA variation to delimit species in a poorly known beetle radiation in the genus Rivacindela from arid Australia. Among 468 individuals sampled from 65 sites and multiple morphologically distinguishable types, sequence variation in three mtDNA genes (cytochrome oxidase subunit 1, cytochrome b, 16S ribosomal RNA) was strongly partitioned between 46 or 47 putative species identified with quantitative methods of species recognition based on fixed unique ("diagnostic") characters. The boundaries between groups were also recognizable from a striking increase in branching rate in clock-constrained calibrated trees. Models of stochastic lineage growth (Yule models) were combined with coalescence theory to develop a new likelihood method that determines the point of transition from species-level (speciation and extinction) to population-level (coalescence) evolutionary processes. Fitting the location of the switches from speciation to coalescent nodes on the ultrametric tree of Rivacindela produced a transition in branching rate occurring at 0.43 Mya, leading to an estimate of 48 putative species (confidence interval for the threshold ranging from 47 to 51 clusters within 2 logL units). Entities delimited in this way exhibited biological properties of traditionally defined species, showing coherence of geographic ranges, broad congruence with morphologically recognized species, and levels of sequence divergence typical for closely related species of insects. The finding of discontinuous evolutionary groupings that are readily apparent in patterns of sequence variation permits largely automated species delineation from DNA surveys of local communities as a scaffold for taxonomy in this poorly known insect group.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                29 August 2012
                : 7
                : 8
                : e43479
                Affiliations
                [1 ]Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
                [2 ]Department of Bioinformatics, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
                [3 ]State Key Laboratory for Infectious Diseases Prevention and Control, Beijing, China
                [4 ]Institute of Zoology, Chinese Academy of Sciences, Beijing, China
                [5 ]QingHai Institute for Endemic Disease Prevention and Control, Xining, Qinghai, China
                University of Vermont, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: LL CZ QL. Performed the experiments: GL YM HM. Analyzed the data: LL DC WZ. Contributed reagents/materials/analysis tools: GL XS HW FM. Wrote the paper: LL DC.

                Article
                PONE-D-12-03414
                10.1371/journal.pone.0043479
                3430698
                22952689
                d28f622e-e93a-4a23-acc4-94c6cff674f5
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 February 2012
                : 20 July 2012
                Page count
                Pages: 13
                Funding
                This work was supported by the Special Infectious Disease Program (Grant No. 2008ZX10004-010) and the National Science and Technology Pillar Program (2008BAI56B02) of the Ministry of Science and Technology, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Computational Biology
                Genomics
                Genome Analysis Tools
                Genetic Screens
                Ecology
                Ecological Metrics
                Species Diversity
                Evolutionary Biology
                Evolutionary Systematics
                Taxonomy
                Animal Taxonomy
                Phylogenetics
                Zoology
                Animal Taxonomy
                Veterinary Science
                Animal Types
                Small Animals

                Uncategorized
                Uncategorized

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