Speciation is often viewed as a continuum along which populations diverge until they become reproductively-isolated species. However, such divergence may be heterogeneous, proceeding in `fits and bursts', rather than being uniform and gradual. We show in Timema stick insects that one form of reproductive isolation indeed evolves non-uniformly across this continuum, whereas another does not. Specifically, we use thousands of host-preference and mating trials to study habitat and sexual isolation among 42 pairs of taxa spanning a range of genomic differentiation and divergence time. We find that the evolution of habitat isolation is uncoupled from genomic differentiation within species, but accumulates linearly with it between species. In contrast, sexual isolation accumulates linearly across the speciation continuum. The results show different evolutionary dynamics for different components of reproductive isolation, indicate sudden transitions between phases of speciation, and highlight a special relevance for species status in the evolution of reproductive isolation.

Host preference was assayed using previously published protocols. Each evening, individual stick insects were placed in a 500-milliliter plastic cup with ~15 cm cuttings of two different host-plant species. The cups were covered with mosquito mesh and left overnight. In the morning we scored which of the two plant species the stick insect was resting upon (trials where neither was chosen were excluded). These experiments tested field-caught individuals within a few days of collection. Habitat isolation was estimated as the degree of divergence between taxon pairs in host-plant feeding preference. This was estimated by assigning one of the two host species used in each assay as the `reference host' and then calculating the absolute value of: (proportion of trials in which taxon 1 of the pair picked the reference host) - (the proportion of trials in which taxon 2 of the pair picked the reference host). Note that the assignment of a reference host is totally arbitrary as the degree of divergence in preference is identical no matter which host species is picked as the reference. 

Mating preference was assayed using previously published protocols. One adult male and one adult female were placed in a standard 10 cm petri dish. Each pair was observed for one hour and scored as having mated or not (Timema have an extended copulation period that is unambiguous and easy to observe). Trials were conducted using the four mating combinations possible for each population pair (male population 1 x female population 1, male population 2 x female population 2; between-population trials: male population 1 x female population 2, male population 2 x female population 1). The same protocol was applied to within- and between-species trials. These experiments largely tested field-caught individuals that were captured as virgin sub-adults and reared to sexual-maturity on the host upon which they were collected, in plastic containers where the sexes were kept separate to ensure virgin status of the individuals tested.  Using the results of these mating trials, we quantified sexual isolation as one minus the frequency of matings for heterospecific (or between population) divided by the frequency of matings for homospecific (or within population) pairs, respectively. 

We obtained nuclear DNA sequence for an anonymous nuclear gene, Tc_nuc235 using primer pairs developed for T. cristinae: Tc_nuc235F = ATCCTGGAATTCACGCACTTAC and Tc_nuc235R = CTTACCCTTCTCCAAAATGTCG. DNA sequences were then obtained using Sanger sequencing, and trimmed and edited to retain 500 bps of high-quality data.

Phylogenetic divergence time estimates were extracted from a published, time-calibrated Bayesian phylogenetic tree (see https://doi.org/10.5061/dryad.nq67q). Demographic inferences were based on a re-alignment and analysis of the same previously published genotyping-by-sequencing data.