Natural and Anthropogenic Hybridization in Two Species of Eastern Brazilian Marmosets (<i>Callithrix jacchus</i> and <i>C</i>. <i>penicillata</i>)

<div><p>Animal hybridization is well documented, but evolutionary outcomes and conservation priorities often differ for natural and anthropogenic hybrids. Among primates, an order with many endangered species, the two contexts can be hard to disentangle from one another, which carries important conservation implications. <i>Callithrix</i> marmosets give us a unique glimpse of genetic hybridization effects under distinct natural and human-induced contexts. Here, we use a 44 autosomal microsatellite marker panel to examine genome-wide admixture levels and introgression at a natural <i>C</i>. <i>jacchus</i> and <i>C</i>. <i>penicillata</i> species border along the São Francisco River in NE Brazil and in an area of Rio de Janeiro state where humans introduced these species exotically. Additionally, we describe for the first time autosomal genetic diversity in wild <i>C</i>. <i>penicillata</i> and expand previous <i>C</i>. <i>jacchus</i> genetic data. We characterize admixture within the natural zone as bimodal where hybrid ancestry is biased toward one parental species or the other. We also show evidence that São Francisco River islands are gateways for bidirectional gene flow across the species border. In the anthropogenic zone, marmosets essentially form a hybrid swarm with intermediate levels of admixture, likely from the absence of strong physical barriers to interspecific breeding. Our data show that while hybridization can occur naturally, the presence of physical, even if leaky, barriers to hybridization is important for maintaining species genetic integrity. Thus, we suggest further study of hybridization under different contexts to set well informed conservation guidelines for hybrid populations that often fit somewhere between “natural” and “man-made.”</p></div

Validation of qPCR Methods for the Detection of <i>Mycobacterium</i> in New World Animal Reservoirs

<div><p>Zoonotic pathogens that cause leprosy (<i>Mycobacterium leprae</i>) and tuberculosis (<i>Mycobacterium tuberculosis</i> complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with <i>M</i>. <i>leprae</i>, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect <i>M</i>. <i>leprae</i> as well as MTBC in wild marmosets. All marmosets were negative for <i>M</i>. <i>leprae</i> DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.</p></div

Brazilian <i>Callithrix jacchus</i> and <i>C</i>. <i>penicillata</i> ranges and sampling locations of parental and hybrid populations.

<p>Orange and light blue areas represent <i>C</i>. <i>jacchus</i> and <i>C</i>. <i>penicillata</i> ranges, respectively, based on 2014 IUCN Red List Spatial Data (<a href="http://www.iucnredlist.org/technical-documents/spatial-data" target="_blank">http://www.iucnredlist.org/technical-documents/spatial-data</a>). Thatched grey suggests region of <i>C</i>. <i>penicillata</i> presence based on Rylands <i>et al</i>. (1993, 2009) and our observations from this study. Degrees of longitude and latitude are, respectively, represented by the <i>x</i>- and the <i>y</i>-axes.</p

Admixture plots resulting from cluster analysis.

<p>A) Plots of <i>C</i>. <i>jacchus</i> and <i>C</i>. <i>penicillata</i> admixture within the two hybrid zones as assigned by STRUCTURE. The two hybrid zones are labeled by their initials. B) Plots of <i>C</i>. <i>jacchus</i> and <i>C</i>. <i>penicillata</i> BAPS admixture probabilities. Plots are divided by reference <i>C</i>. <i>penicillata</i> individuals (P), reference <i>C</i>. <i>jacchus</i> (J), and the hybrid zones labeled by their initials. In both plots, purple and green bar proportions indicate <i>C</i>. <i>penicillata</i> and <i>C</i>. <i>jacchus</i> ancestry, respectively. Within the PJ and RJ panels, black lines separate individual capture sites within each hybrid zone, following the order given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127268#pone.0127268.g002" target="_blank">Fig 2</a> for the PJ zone and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127268#pone.0127268.g003" target="_blank">Fig 3</a> for the RJ zone. White lines within each hybrid zone panel separate the southern and northern portions of each hybrid zone. Also, in panel A, CEMAFAUNA captive marmosets are found between the two white lines.</p

Distribution of mycobacteria identified in case study.

<p>Geographic and taxonomic distribution of rpoB1 positive marmoset samples. rpoB1 positive samples are found in Recife, Pernambuco (n = 4), the neighboring municipalities of Petrolina, Pernambuco and Juazeiro, Bahia (n = 1), and the neighboring cities of Silva Jardim and Rio Bonito, Rio de Janeiro (n = 9) and in <i>C</i>. <i>jacchus</i> (n = 3), hybrids (n = 10), and <i>C</i>. <i>penicillata</i> (n = 1).</p

PCA of microsatellite allele frequencies.

<p>Plot of first and second components from the PCA showing genetic differences in terms of microsatellite multilocus genotypes between marmoset parental species and hybrid zone samples. Individual <i>C</i>. <i>jacchus</i> are colored in green, individual <i>C</i>. <i>penicillata</i> are colored in purple, and individuals from the PJ and RJ hybrid zones, respectively, are colored blue and orange.</p

Overall qPCR results for case study.

<p>qPCR results for marmoset samples (n = 98). All marmosets were negative for the <i>M</i>. <i>leprae</i>-specific 85B and rlep genes and the MTBC-specific rpoB2 and IS6110 genes. However, 14 are positive for the mycobacterial rpoB1 gene. Approximately 0.001–0.137 mycobacterial genome copies per ng of DNA extracted were identified from these rpoB1-positive samples.</p

Goodness of fit analysis for trial study.

<p>ELISA and qPCR test results for the armadillo trial study as compared to the expected results based on known armadillo experimental infection statuses (infected n = 25, non-infected n = 20). Each assay type is compared to the expected using Chi-squared analyses. Assay types include the PGL1 ELISA (infected n = 11, non-infected n = 34, χ2 = 17.52, p<0.01), LID1 ELISA (infected n = 15, non-infected n = 30, χ2 = 8.92, p<0.01), 85B qPCR (infected n = 9, non-infected n = 36, χ2 = 22.90, p<0.01), rlep qPCR (infected n = 26, non-infected n = 19, χ2 = 0.12, p = 0.73), and combination of 85B and rlep qPCRs (85B+rlep, infected n = 18, non-infected n = 27, χ2 = 4.36, p = 0.04). Chi-squared analyses show all tests significantly differ from the expected (*p<0.01) except the qPCR tests performed using only the rlep target and the combination of 85B and rlep.</p

Detail of Rio de Janeiro State anthropogenic hybrid zone.

<p>We sampled within the zone along an approximately 30 km transect paralleling highway BR-101. Four sites were found to the south of the highway: (1) Boa Esperança; (2) House U; (3) Rio Vermelho I; and (4) Rio Vermelho II. Four sites were found to the north of the highway: (5) Fazenda dos Tamarins; (6) Pesque Pague; (7) Ponto do Camarão; and (8) Fazenda Afetiva.</p

Detail of the Petrolina-Juazeiro natural hybrid zone.

<p>We sampled within the hybrid zone along an approximately 50 km transect paralleling the São Francisco River. Three sites were found to the south of the river: (1) Universidade do Estado da Bahia; (2) Chácara do Senhor Conrado dos Santos; and (3) Recanto do Sessego. Six sites were found to the north of the river: (4) Sítio Porto da Cruz; (5) Rio Verde; (6) Sítio Picos; (7) Sítio Carnaíba; (8) Chácara Galo da Briga; and (9) Chácara Bom Jesus.</p