Museum of Southwestern Biology and Department of Biology, MSC03 2020, 1 University of New Mexico, Albuquerque, New Mexico.
Department of Biology, University of Nevada, Reno, Nevada.
Mol Ecol Resour. 2019 Mar;19(2):400-410. doi: 10.1111/1755-0998.12977.
Avian malaria and related haemosporidians (Plasmodium, [Para]Haemoproteus and Leucocytoozoon) represent an exciting multihost, multiparasite system in ecology and evolution. Global research in this field accelerated after the publication in 2000 of PCR protocols to sequence a haemosporidian mitochondrial (mtDNA) barcode and the development in 2009 of an open-access database to document the geographic and host ranges of parasite mtDNA haplotypes. Isolating haemosporidian nuclear DNA from bird hosts, however, has been technically challenging, slowing the transition to genomic-scale sequencing techniques. We extend a recently developed sequence capture method to obtain hundreds of haemosporidian nuclear loci from wild bird samples, which typically have low levels of infection, or parasitemia. We tested 51 infected birds from Peru and New Mexico and evaluated locus recovery in light of variation in parasitemia, divergence from reference sequences and pooling strategies. Our method was successful for samples with parasitemia as low as ~0.02% (2 of 10,000 blood cells infected) and mtDNA divergence as high as 15.9% (one Leucocytozoonsample), and using the most cost-effective pooling strategy tested. Phylogenetic relationships estimated with >300 nuclear loci were well resolved, providing substantial improvement over the mtDNA barcode. We provide protocols for sample preparation and sequence capture including custom probe sequences and describe our bioinformatics pipeline using atram 2.0, phyluce and custom Perl/Python scripts. This approach can be applied to thousands of avian samples that have already been found to have haemosporidian infections of at least moderate intensity, greatly improving our understanding of parasite speciation, biogeography and evolutionary dynamics.
鸟类疟疾和相关血孢子虫（疟原虫、[副]疟血虫和白细胞寄生菌）在生态学和进化方面代表了一个令人兴奋的多宿主、多寄生虫系统。2000 年发表了用于测序血孢子虫线粒体（mtDNA）条形码的 PCR 方案，2009 年开发了一个开放获取数据库来记录寄生虫 mtDNA 单倍型的地理和宿主范围后，该领域的全球研究加速了。然而，从鸟类宿主中分离血孢子虫核 DNA 在技术上具有挑战性，这减缓了向基因组规模测序技术的转变。我们扩展了最近开发的序列捕获方法，从野生鸟类样本中获得数百个血孢子虫核基因座，这些样本通常感染水平低或寄生率低。我们测试了来自秘鲁和新墨西哥的 51 只感染的鸟类，并根据寄生率、与参考序列的差异和混合策略评估了基因座的恢复情况。我们的方法对寄生率低至约 0.02%（10000 个血细胞中感染了 2 个）和 mtDNA 差异高达 15.9%（一个白细胞寄生菌样本）的样本是成功的，并且使用了测试的最具成本效益的混合策略。用>300 个核基因座估计的系统发育关系得到了很好的解决，与 mtDNA 条形码相比有了很大的改进。我们提供了样本制备和序列捕获的方案，包括定制探针序列，并描述了我们使用 atram 2.0、phyluce 和自定义 Perl/Python 脚本的生物信息学管道。这种方法可以应用于已经发现至少中度强度血孢子虫感染的数千个鸟类样本，这极大地提高了我们对寄生虫物种形成、生物地理学和进化动态的理解。
