Hybrid cline or hybrid lineage: A genomic reevaluation of Sibley’s classic species conundrum in Pipilo towhees

Hybrid zones are often described as clines of genetic and phenotypic traits moving across species barriers through introgression. Yet, hybrid zones can also be spatially complex and shift over time, and dispersal and vicariance can isolate portions of a cline, potentially leading to hybrid lineage formation. We reassessed Sibley’s (1950) gradient between Collared Towhee (Pipilo ocai) and Spotted Towhee (P. maculatus) in Central Mexico to test whether it conformed to a typical tension-zone cline model. By comparing historical and modern data, we found that cline centers for genetic and phenotypic traits have not shifted over the course of 70 years. This equilibrium suggests that secondary contact between these species, which originally diverged over 2 million years ago, likely dates to the Pleistocene. Given the amount of mtDNA divergence, parental ends of the cline have very low autosomal nuclear differentiation (FST = 0.12). Dramatic and coincident cline shifts in mtDNA and throat color suggest the possibility of sexual selection as a factor in differential introgression, while a contrasting cline shift in green back color hints at a role for natural selection. Supporting the idea of a continuum between hybrid clines and hybrid lineage formation, the towhee gradient can be analyzed as one population under isolation-by-distance, as a two-population cline, and as three lineages experiencing divergence with gene flow. In the middle of the gradient, a hybrid lineage has become partly isolated, likely due both to forested habitat shrinking and fragmenting as it moved upslope after the last glacial maximum and a stark environmental transition. The towhee system offers a window into the potential outcomes of hybridization across a dynamic landscape including the creation of novel genomic and phenotypic combinations and incipient hybrid lineages.

Methods
Morphological data: traits measured from specimens with ruler and digital calipers

Genetic data: SNPs generated from sequence capture of ultraconserved elements and Illumina sequencing

Environmental data: extracted from environmental layers from localities associated with photo vouchers on iNaturalist

杂交带（hybrid zone）常被描述为通过基因渐渗（introgression）跨越物种壁垒的遗传与表型性状渐变群（cline）。然而，杂交带也可具有空间复杂性并随时间推移发生位移，扩散与地理分异（vicariance）事件可隔离渐变群的部分区域，进而可能催生杂交谱系的形成。

我们重新评估了1950年Sibley提出的墨西哥中部领唧鹀（Collared Towhee，学名*Pipilo ocai*）与斑唧鹀（Spotted Towhee，学名*P. maculatus*）之间的性状渐变群，以验证其是否符合典型的张力区渐变群（tension-zone cline）模型。通过对比历史与现代数据，我们发现遗传与表型性状的渐变群中心在70年间未发生位移。这种平衡状态表明，这两种原本在200余万年前发生分化的物种之间的二次接触（secondary contact），大概率发生于更新世（Pleistocene）时期。

考虑到线粒体DNA（mitochondrial DNA, mtDNA）的分化程度，渐变群的两个亲本端的常染色体核分化程度极低（遗传分化系数FST=0.12）。线粒体DNA与喉部羽色的渐变群发生显著且同步的位移，提示性选择可能在差异性基因渐渗中发挥作用；而背部绿色调的渐变群位移则与之相反，暗示自然选择的影响。

为支持杂交渐变群与杂交谱系形成之间存在连续性这一观点，唧鹀的性状渐变群可被分析为受距离隔离（isolation-by-distance）影响的单一种群、双种群渐变群，以及存在基因流的三个分化谱系。在渐变群的中部，一个杂交谱系已发生部分隔离，其成因可能是末次冰盛期后栖息地随海拔抬升发生的森林退缩与碎片化，以及显著的环境转变。该唧鹀研究系统为解析动态景观中杂交的潜在结局提供了研究窗口，包括全新基因组与表型组合的产生，以及处于形成初期的杂交谱系。

研究方法
形态学数据：采用直尺与数显游标卡尺对标本的性状进行测量
遗传学数据：通过超保守元件（ultraconserved elements）序列捕获与Illumina测序生成单核苷酸多态性（Single Nucleotide Polymorphism, SNP）标记
环境数据：从iNaturalist平台上与附有照片凭证的采样点对应的环境图层中提取数据