C. William Kilpatrick

Multiple molecular evidences for a living mammalian fossil

Laonastes aenigmamus is an enigmatic rodent first described in 2005. Molecular and morphological data suggested that it is the sole representative of a new mammalian family, the Laonastidae, and a member of the Hystricognathi. However, the validity of this family is controversial because fossil-based phylogenetic analyses suggest that Laonastes is a surviving member of the Diatomyidae, a family considered to have been extinct for 11 million years. According to these data, Laonastes and Diatomyidae are the sister clade of extant Ctenodactylidae (i.e., gundies) and do not belong to the Hystricognathi. To solve the phylogenetic position of Laonastes, we conducted a large-scale molecular phylogeny of rodents. The analysis includes representatives of all major rodent taxonomic groups and was based on 5.5 kb of sequence data from four nuclear and two mitochondrial genes. To further validate the obtained results, a short interspersed element insertion analysis including 11 informative loci was also performed. Our molecular data based on sequence and short interspersed element analyses unambiguously placed Laonastes as a sister clade of gundies. All alternative hypotheses were significantly rejected based on Shimodaira–Hasegawa tests, supporting the idea that Laonastes does not belong to the Hystricognathi. Molecular dating analysis also supports an ancient divergence, ≈44 Mya ago, between Ctenodactylidae and Laonastes. These combined analyses support the hypothesis that Laonastes is indeed a living fossil. Protection of this surviving species would conserve an ancient mammalian family.

Morphological and molecular investigations of a new family, genus and species of rodent (Mammalia: Rodentia: Hystricognatha) from Lao PDR

Abstract During biodiversity surveys in Khammouan Province, Lao Peoples Democratic Republic, specimens of an unknown species of hystricognathous rodent were discovered in local markets being sold for food; local hunters explaining that these rock rats were trapped in the nearby limestone karst. These specimens are described here on the basis of their unique combination of external and craniodental features as members of a new family, genus and species, using comparative morphological and molecular data. Phylogenetic analyses of morphological data and of 12S rRNA and cytochrome b are presented on selected taxa from all suborders of Rodentia. The results of the molecular and morphological analyses are compared and provide the basis for a discussion of relationships of the new taxon within the Rodentia and Hystricognatha. The disjunct distribution of hystricognaths is recognised as problematic, with most families occurring in the main distributional area of South America, several others in Africa and only one family distributed in Africa and Asia. The presence of an additional hystricognath family in Southeast Asia poses interesting questions and consideration is given to the way in which this new taxon fits into the theories of the biogeographical and evolutionary history of other hystricognaths. Features of possible ecomorphological significance are briefly discussed, such as the apparent adaptations to a rock dwelling existence evident in various features of the external morphology and comparisons are made to the morphology of other known saxicolous rodents.

Toward a Molecular Phylogeny for Peromyscus: Evidence from Mitochondrial Cytochrome-b Sequences

Abstract One hundred DNA sequences from the mitochondrial cytochrome-b gene of 44 species of deer mice (Peromyscus (sensu stricto), 1 of Habromys, 1 of Isthmomys, 2 of Megadontomys, and the monotypic genera Neotomodon, Osgoodomys, and Podomys were used to develop a molecular phylogeny for Peromyscus. Phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference) were conducted to evaluate alternative hypotheses concerning taxonomic arrangements (sensu stricto versus sensu lato) of the genus. In all analyses, monophyletic clades were obtained that corresponded to species groups proposed by previous authors; however, relationships among species groups generally were poorly resolved. The concept of the genus Peromyscus based on molecular data differed significantly from the most current taxonomic arrangement. Maximum-likelihood and Bayesian trees depicted strong support for a clade placing Habromys, Megadontomys, Neotomodon, Osgoodomys, and Podomys within Peromyscus. If Habromys, Megadontomys, Neotomodon, Osgoodomys, and Podomys are regarded as genera, then several species groups within Peromyscus (sensu stricto) should be elevated to generic rank. Isthmomys was associated with the genus Reithrodontomys; in turn this clade was sister to Baiomys, indicating a distant relationship of Isthmomys to Peromyscus. A formal taxonomic revision awaits synthesis of additional sequence data from nuclear markers together with inclusion of available allozymic and karyotypic data.

Noncryogenic Preservation of Mammalian Tissues for DNA Extraction: An Assessment of Storage Methods

Reliable field methods for the storage of tissues to be used for DNA extraction and amplification are critical to many studies employing molecular techniques. Protection from DNA degradation was compared among three commonly used methods of noncryogenic storage of tissues over a time scale of 2 years. All three methods prevented DNA degradation during storage for at least 6 months. DMSO (dimethyl sulfoxide)-salt solution provided the best protection from DNA degradation of tissues stored for up to 2 years. High molecular weight DNA was recovered from lysis buffer in which tissue was stored for 2 years, however, moderate amounts of degraded DNA was also present. High molecular weight DNA was recovered from tissues stored in ethanol for 2 years, however, the yield was relatively small compared to the other two noncryogenic storage techniques. Much of the DNA degradation in ethanol preserved tissues appeared to occur during the extraction procedure and can be reduced by soaking the tissue in lysis buffer for a few hours prior to beginning the extraction. The yield of PCR products was greatest from DNA extracted from DMSO-salt solution preserved tissues, whereas DNA from tissues stored in either lysis buffer or ethanol produced lower yields.

Morphological Differentiation and Identification of Peromyscus leucopus and P. maniculatus in Northeastern North America

External features of the tail and pelage, and quantitative cranial characteristics were used to discriminate Peromyscus leucopus from P. maniculatus ( n = 204) from northeastern North America. Species assignments were based on the phenotype of salivary amylase. Characteristics of the pelage and tail yielded correct identification of 55% of adult specimens. A previously published discriminant-function equation based on 11 cranial measurements correctly classified 66% of adults and 56% of specimens of all age classes. Two new discriminant equations were generated based on 12 and 11 skull measurements, respectively. The first equation correctly classified 100% of skulls in two separate datasets ( n = 164; n = 50), and the second correctly classified 94% in a single dataset ( n = 195).

A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

A NEW SPECIES OF PEROMYSCUS FROM WESTERN MEXICO

Abstract Historically, specimens representing the Peromyscus boylii species group from west-central Mexico have been referred to as Peromyscus boylii; however, a distinct but polymorphic karyotype (fundamental number 54–56) precludes an assignment of these specimens to currently recognized taxa. Phylogenetic analyses (parsimony, likelihood, and Bayesian) of DNA sequences indicated that samples from Durango, Mexico, formed a monophyletic clade that either was sister to P. levipes (parsimony) or to a clade containing P. beatae and P. levipes (likelihood and Bayesian). To refer these samples to P. boylii results in paraphyly and a significantly worse topology. Together, these results indicate that the samples from the Sierra Madre Occidental region of Durango and southeastern Sinaloa represent an undescribed species of Peromyscus. This taxon is described herein as a new species.

TAXONOMIC STATUS OF PEROMYSCUS BOYLII SACARENSIS: INFERENCES FROM DNA SEQUENCES OF THE MITOCHONDRIAL CYTOCHROME-B GENE

Abstract Taxonomic affinity of Peromyscus boylii sacarensis was examined using DNA sequences from the mitochondrial cytochrome-b gene. Maximum parsimony and genetic distance analyses were used to examine the phylogenetic relationship of this taxon to other closely related members of the P. boylii species group (P. beatae, P. boylii rowleyi, P. levipes ambiguus, P. l. levipes, and P. simulus). All analyses, despite differential weighting of transitions and nucleotide positions (1st, 2nd, and 3rd) or method of calculating genetic distances, resulted in similar topologies with P. b. sacarensis forming the sister group to P. beatae. Our data suggest that P. b. sacarensis should be aligned taxonomically with P. beatae instead of its current placement in P. boylii and that 3 genetic subunits exist within P. beatae.

PHYLOGENETIC RELATIONSHIPS OF NEOTOMINE–PEROMYSCINE RODENTS: BASED ON DNA SEQUENCES FROM THE MITOCHONDRIAL CYTOCHROME-b GENE

Abstract DNA sequences obtained from the mitochondrial cytochrome-b gene were used to evaluate phylogenetic relationships among 15 genera of putative neotomine–peromyscine rodents. Tree topologies obtained from maximum likelihood and Bayesian analyses revealed 4 primary clades that, in general, conform to the 4 tribes proposed by Carleton (1980). The Peromyscini (Megadontomys, Ochrotomys, Osgoodomys, Peromyscus, and Reithrodontomys) was sister to the Neotomini (Hodomys, Neotoma, Onychomys, and Xenomys). These 2 clades were then joined by the Baiomyini (Baiomys and Scotinomys) and Tylomyini (Nyctomys, Ototylomys, and Tylomys). The most apparent departure from previously proposed arrangements involved the placement of Onychomys in the Neotomini instead of the Peromyscini.

Phylogeographic study of Apodemus ilex (Rodentia: Muridae) in Southwest China.

Background The Mountains of southwest China have complex river systems and a profoundly complex topography and are among the most important biodiversity hotspots in the world. However, only a few studies have shed light on how the mountains and river valleys promote genetic diversity. Apodemus ilex is a fine model for investigating this subject. Methodology/Principal Findings To assess the genetic diversity and biogeographic patterns of Apodemus ilex, the complete cytochrome b gene sequences (1,140 bp) were determined from 203 samples of A. draco/ilex that were collected from southwest China. The results obtained suggested that A. ilex and A. draco are sistergroups and diverged from each other approximately 2.25 million years ago. A. ilex could be divided into Eastern and Western phylogroups, each containing two sub-groups and being widespread in different geographical regions of the southern Hengduan Mountains and the western Yunnan - Guizhou Plateau. The population expansions of A. ilex were roughly from 0.089 Mya to 0.023 Mya. Conclusions Our result suggested that A. ilex is a valid species rather than synonym of A. draco. As a middle-high elevation inhabitant, the phylogenetic pattern of A. ilex was strongly related to the complex geographical structures in southwest China, particularly the existence of deep river valley systems, such as the Mekong and Salween rivers. Also, it appears that the evolutionary history of A. ilex, such as lineage divergences and population expansions were strongly affected by climate fluctuation in the Late Pleistocene.