Intra-specific Y-chromosomal sequence variation is useful for analysing the male
contribution to a species’ spatial genetic structure. In red deer (Cervus elaphus) this
is especially relevant, because geographic dispersal and game translocations occur
mainly through the males. However, Y-chromosomal markers for wild organisms are
scarce and frequently non-polymorphic within species. We assessed the intra-specific
variation of two Y-chromosomal introns in red deer, one in the DBY (or DDX3Y) gene
and the other in the UBE1Y gene. The introns were amplified using previously published
exonic primers and directly sequenced in individuals of five red deer subspecies
from across Eurasia. However, no nucleotide polymorphism was observed, which
rebuts the usefulness of these introns for studies of red deer phylogeography and on
illegal transport of red deer within this region. Male-based phylogeographic studies
should thus be focused on other Y-chromosomal markers for this species

Barbosa, A. Márcia

Carranza, J.

Repositório Científico da Universidade de Évora

Journal of Negative Results— Ecology & Evolutionary Biology —vol. 7: 1–4 • Helsinki, 26 March 2010ISSN 1459-4625 http://www.jnr-eeb.org/Editor in charge of this manuscript: Bob O’HaraLack of geographic variation in Y-chromosomal introns of red deer (Cervus elaphus)A. Márcia Barbosa1,2,3,* and Juan Carranza1,41) Biología Evolutiva, Etología y Gestión Cinegética, Facultad de Veterinaria, Universidad de Extremadura, 10071 Cáceres, Spain2) Cátedra “Rui Nabeiro” — Biodiversidade, CIBIO — Universidade de Évora, Largo dos Colegiais, 7004-516 Évora, Portugal (corresponding author’s e-mail: barbosa@uevora.pt)3) Department of Biological Sciences, Imperial College London, Silwood Park Campus, Ascot SL5 7PY, United Kingdom4) Ungulate Research Unit, CRCP, Universidad de Córdoba, 14071 Córdoba, SpainReceived 17 December 2009 accepted 16 March 2010Intra-specific Y-chromosomal sequence variation is useful for analysing the male contribution to a species’ spatial genetic structure. In red deer (Cervus elaphus) this is especially relevant, because geographic dispersal and game translocations occur mainly through the males. However, Y-chromosomal markers for wild organisms are scarce and frequently non-polymorphic within species. We assessed the intra-specific variation of two Y-chromosomal introns in red deer, one in the DBY (or DDX3Y) gene and the other in the UBE1Y gene. The introns were amplified using previously pub-lished exonic primers and directly sequenced in individuals of five red deer subspecies from across Eurasia. However, no nucleotide polymorphism was observed, which rebuts the usefulness of these introns for studies of red deer phylogeography and on illegal transport of red deer within this region. Male-based phylogeographic studies should thus be focused on other Y-chromosomal markers for this species.IntroductionAs the only exclusively male-transmitted part of the mammalian genome, the non-recombining portion of the Y chromosome is an invaluable tool for studying the male contribution to a spe-cies’ genetic structure. However, the mammalian Y chromosome generally has low nucleotide diversity (Hellborg & Ellegren 2004), hamper-ing male-based phylogeographic studies. None-theless, Hurles and Jobling (2001) and Petit et al. (2002) recommended using male-specific genetic markers, which can be informative for several species (e.g. Cathey et al. 1998, Under-hill et al. 2000, Geraldes et al. 2005).In a species such as the red deer (Cervus elaphus, Cetartiodactyla: Cervidae), male-based phylogeographic studies can be especially rel-evant, because geographic dispersal occurs mainly through males (Clutton-Brock et al. 1982, 2002). Furthermore, the red deer is an economically important game species which is hunted mainly for antler trophies, hence illegal transport of male animals for game purposes also occur (Carranza et al. 2003).Hellborg and Ellegren (2003) designed exonic primers for a series of mammalian Y-chromosomal introns. We analysed two primer pairs that achieved amplification in the two cervid species tested by these authors, reindeer JNR EEB vol. 7 • Barbosa & Carranza: Lack of deer Y-chromosomal intron variation 2(Rangifer tarandus) and roe deer (Capreolus capreolus). The target introns have not shown polymorphism in every species analysed so far (e.g. Hellborg & Ellegren 2004). However, red deer show considerable intraspecific diversity at other regions of their genome, such as the mitochondrial DNA control region (D-loop), cytochrome b, and nuclear microsatellites (e.g., Feulner et al. 2004, Ludt et al. 2004, Skog et al. 2009). Several red deer subspecies have been described, and there is even support for a subdi-vision into two species (Ludt et al. 2004) within Eurasia. We therefore expected Y-chromosomal introns to exhibit variability among scattered Eurasian populations of this species. The aim of this study was, thus, to gauge the variability of these introns among several red deer popula-tions, in order to assess their usefulness for male-based phylogeograhic studies on this species.Material and methodsBiological samples consisted of antlers or flesh of male individuals of five red deer subspecies coming from five different countries (Table 1), altogether spanning a substantial part of the Palearctic region (Fig. 1). Only one individual from each subspecies was sampled, as the aim was to check if there could be enough variation to perform a phylogeographic study based on the analysed markers.Total DNA was extracted using a salt-out pro-cedure (Miller et al. 1988) on flesh pieces or antler shavings. The target introns were then amplified by polymerase chain reaction (PCR) using the primer pairs DBY8 (forward: CCCCAACAAGA-GAATTGGCT, reverse: CAG CA CCA CCA TAK-ACTACA) and UBE1Y6 (forward: CCCCTGCA-GACCKRCAT, reverse: AAGGCCAAGTTGAT-RAARCT) designed by Hellborg and Ellegren (2003). PCR products were electrophoresed on 1.6% agarose gels stained with SYBR safe (Invit-rogen, Barcelona) and visualized under ultraviolet light to confirm amplification and assess the size of the fragments.Amplicons were purified using ExoSAP-IT (GE Healthcare) and directly sequenced in both directions using the PCR primers and a BigDye Terminator v1.1 Cycle Sequencing kit (Applied BioSystems). Sequences were recorded with an ABI3130 automated sequencer and aligned with BioEdit (Hall 1999) for comparison. A FASTA search (Pearson & Lipman 1988) was performed to confirm correspondence with the target genes.Results and discussionThe approximate sizes of the amplified UBE1Y6 and DBY8 introns were 300 and 200 bp, respec-tively. The sequences were uploaded to GenBank and their accession numbers are provided (Table 1). Sequence alignment and comparison revealed no polymorphisms among the analysed individu-als, in neither the DBY8 nor the UBE1Y6 intron.Although the sample size was small, the ana-lysed individuals represent five distinct nomi-nal subspecies (Table 1) and encompass a wide geographical coverage (Fig. 1). The results thus strongly suggest reduced variation at these loci in red deer, at least within Eurasia. Variation might still exist among populations, but the value of these markers for phylogeographic studies of this Table 1. Subspecies of Cervus elaphus analysed, geographical origin of their samples, and the Gen-Bank accession numbers of the nucleotide sequences obtained from each of the two analysed introns.Subspecies Origin UBE1Y6 DBY8C. e. hispanicus Spain EU219368 EU219373C. e. atlanticus Norway EU219369 EU219374C. e. scoticus Scotland EU219370 EU219375C. e. hippelaphus Hungary EU219371 EU219376C. e. maral Iran EU219372 EU219377Fig. 1. Geographical origins of the analysed red deer samples.JNR EEB vol. 7 • Barbosa & Carranza: Lack of deer Y-chromosomal intron variation 3species is visibly limited. This lack of variation, however, is not generalised in the genome of the analysed red deer: the same individuals studied here show nucleotide polymorphisms at mitochon-drial loci (J. L. Fernández-García unpubl. data).Hellborg and Ellegren (2004) showed that intraspecific Y-chromosome variability differs significantly between species. The same introns we analysed have shown intra-specific variabil-ity in wolf Canis lupus and field vole Microtus agrestis (Hellborg & Ellegren 2004, Hellborg et al. 2005), for example. Conversely, Brändli et al. (2005) found significant intraspecific variation in the Y chromosome of greater white-toothed shrew Crocidura russula, but not in the DBY8 intron, which they also analysed. Our introns have also shown absence of variability within reindeer Rangifer tarandus, cattle Bos taurus (Hellborg & Ellegren 2004) and the horse Equus caballus (Lindgren et al. 2005).Other studies have revealed low general Y-chromosomal variability, both within wild species such as the hamadryas baboon Papio hamadryas (Lawson Handley et al. 2006) and within domestic species such as the horse (Lind-gren et al. 2005). This uniformity was attributed to biased reproduction caused by polygyny in the former case, and by sex-biased domestication in the latter, with few males contributing geneti-cally to the subsequent generations. The fact that red deer also have a polygynous mating system (Clutton-Brock et al. 1982) may, therefore, help to explain the low variability observed in the analysed Y-chromosomal sequences.Despite the high intraspecific genetic diver-sity observed elsewhere in the red deer genome (e.g., Ludt et al. 2004, Skog et al. 2009, J. L. Fernández-García unpubl. data) and the fact that red deer females appear to mate with genetically dissimilar males (Carranza et al. 2009), nei-ther of the two analysed Y-chromosomal introns exhibited nucleotide differences in any of the five subspecies analysed. Male-based phylogeo-graphic studies on red deer should thus turn to other Y-chromosomal markers.AcknowledgementsWe thank everyone that collected and sent us biological samples. Carmen Galán and Sheila Martín performed part of the amplification and sequencing. Leticia Castillo and Javier Pérez-González provided invaluable help. Funding was provided by the Spanish Ministry of Education and Sci-ence (projects GLC2004-059939 and CGL2007-63594) and FEDER / Junta de Extremadura (project PDT05A043). A. M. B. is currently supported by a fellowship from Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BPD/40387/2007).ReferencesBrändli, L., Lawson Handley, L.-J., Vogel, P. & Perrin, N. 2005: Evolutionary history of the greater white-toothed shrew (Crocidura russula) inferred from analy-sis of mtDNA, Y, and X chromosome markers. — Mol. Phylog. Evol. 37: 832–844.Carranza, J., Martínez, J. G., Sánchez-Prieto, C. B., Fern-ández-García, J. L., Sánchez-Fernández, B., Álvarez–Álvarez, R., Valencia J. & Alarcos, S. 2003: Game species: extinction hidden by census numbers. — Anim. Biodivers. 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Lack of geographic variation in Y-chromosomal introns of red deer (Cervus elaphus)

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Lack of geographic variation in Y-chromosomal introns of red deer (Cervus elaphus)

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