Evolution of sea-surfing plant propagule as revealed by the genomes of Heritiera mangroves.

Coastal forests, such as mangroves, protect much of the tropical and subtropical coasts. Long-distance dispersal via sea-surfing propagules is essential for coastal plants, but the genomic and molecular basis of sea-surfing plant propagule evolution remains unclear. Heritiera fomes and Heritiera littoralis are two coastal plants with typical buoyant fruits. We de novo sequenced and assembled their high-quality genomes. Our phylogenomic analysis indicates H. littoralis and H. fomes originated (at ~6.08 Mya) just before the start of Quaternary sea-level fluctuations. Whole-genome duplication occurred earlier, permitting gene copy gains in the two species. Many of the expanded gene families are involved in lignin and flavonoid biosynthesis, likely contributing to buoyant fruit emergence. It is repeatedly revealed that one duplicated copy to be under positive selection while the other is not. By examining H. littoralis fruits at three different developmental stages, we found that gene expression levels remain stable from young to intermediate. However, ~1000 genes are up-regulated and ~ 3000 genes are down-regulated as moving to mature. Particularly in fruit epicarps, the upregulation of WRKY12 and E2Fc likely constrains the production of p-Coumaroyl-CoA, the key internal substrate for lignin biosynthesis. Hence, to increase fruit impermeability, methylated lignin biosynthesis is shut down by down-regulating the genes CCoAOMT, F5H, COMT, and CSE, while unmethylated lignins are preferentially produced by upregulating CAD and CCR. Similarly, cutin polymers and cuticular waxes accumulate with high levels before maturation in epicarps. Overall, our genome assemblies and analyses uncovered the genomic evolution and temporal transcriptional regulation of sea-surfing propagule.

Adaptation in Unstable Environments and Global Gene Losses: Small but Stable Gene Networks by the May-Wigner Theory.

Although gene loss is common in evolution, it remains unclear whether it is an adaptive process. In a survey of seven major mangrove clades that are woody plants in the intertidal zones of daily environmental perturbations, we noticed that they generally evolved reduced gene numbers. We then focused on the largest clade of Rhizophoreae and observed the continual gene set reduction in each of the eight species. A great majority of gene losses are concentrated on environmental interaction processes, presumably to cope with the constant fluctuations in the tidal environments. Genes of the general processes for woody plants are largely retained. In particular, fewer gene losses are found in physiological traits such as viviparous seeds, high salinity, and high tannin content. Given the broad and continual genome reductions, we propose the May-Wigner theory (MWT) of system stability as a possible mechanism. In MWT, the most effective solution for buffering continual perturbations is to reduce the size of the system (or to weaken the total genic interactions). Mangroves are unique as immovable inhabitants of the compound environments in the land-sea interface, where environmental gradients (such as salinity) fluctuate constantly, often drastically. Extending MWT to gene regulatory network (GRN), computer simulations and transcriptome analyses support the stabilizing effects of smaller gene sets in mangroves vis-à-vis inland plants. In summary, we show the adaptive significance of gene losses in mangrove plants, including the specific role of promoting phenotype innovation and a general role in stabilizing GRN in unstable environments as predicted by MWT.

The evolution history of an allotetraploid mangrove tree analysed with a new tool Allo4D.

Mangrove species are broadly classified as true mangroves and mangrove associates. The latter are amphibious plants that can survive in the intertidal zone and reproduce naturally in terrestrial environments. Their widespread distribution and extensive adaptability make them ideal research materials for exploring adaptive evolution. In this study, we de novo assembled two genomes of mangrove associates (the allotetraploid Barringtonia racemosa (2n = 4x = 52) and diploid Barringtonia asiatica (2n = 2x = 26)) to investigate the role of allopolyploidy in the evolutionary history of mangrove species. We developed a new allotetraploid-dividing tool Allo4D to distinguish between allotetraploid scaffold-scale subgenomes and verified its accuracy and reliability using real and simulated data. According to the two subgenomes of allotetraploid B. racemosa divided using Allo4D, the allopolyploidization event was estimated to have occurred approximately one million years ago (Mya). We found that B. racemosa, B. asiatica, and Diospyros lotus shared a whole genome duplication (WGD) event during the K-Pg (Cretaceous-Paleozoic) period. K-Pg WGD and recent allopolyploidization events contributed to the speciation of B. racemosa and its adaptation to coastal habitats. We found that genes in the glucosinolates (GSLs) pathway, an essential pathway in response to various biotic and abiotic stresses, expanded rapidly in B. racemosa during polyploidization. In summary, this study provides a typical example of the adaptation of allopolyploid plants to extreme environmental conditions. The newly developed tool, Allo4D, can effectively divide allotetraploid subgenomes and explore the evolutionary history of polyploid plants, especially for species whose ancestors are unknown or extinct.

Genomic analysis of Nypa fruticans elucidates its intertidal adaptations and early palm evolution.

Nypa fruticans (Wurmb), a mangrove palm species with origins dating back to the Late Cretaceous period, is a unique species for investigating long-term adaptation strategies to intertidal environments and the early evolution of palms. Here, we present a chromosome-level genome sequence and assembly for N. fruticans. We integrated the genomes of N. fruticans and other palm family members for a comparative genomic analysis, which confirmed that the common ancestor of all palms experienced a whole-genome duplication event around 89 million years ago, shaping the distinctive characteristics observed in this clade. We also inferred a low mutation rate for the N. fruticans genome, which underwent strong purifying selection and evolved slowly, thus contributing to its stability over a long evolutionary period. Moreover, ancient duplicates were preferentially retained, with critical genes having experienced positive selection, enhancing waterlogging tolerance in N. fruticans. Furthermore, we discovered that the pseudogenization of Early Methionine-labelled 1 (EM1) and EM6 in N. fruticans underly its crypto-vivipary characteristics, reflecting its intertidal adaptation. Our study provides valuable genomic insights into the evolutionary history, genome stability, and adaptive evolution of the mangrove palm. Our results also shed light on the long-term adaptation of this species and contribute to our understanding of the evolutionary dynamics in the palm family.

Adaptation to a new environment with pre-adaptive genomic features - Evidence from woody plants colonizing the land-sea interface.

Adaptation to new environments is a key evolutionary process which presumably involves complex genomic changes. Mangroves, a collection of approximately 80 woody plants that have independently invaded intertidal zones >20 times, are ideal for studying this process. We assembled near-chromosome-scale genomes of three Xylocarpus species as well as an outgroup species using single-molecule real-time sequencing. Phylogenomic analysis reveals two separate lineages, one with the mangrove Xylocarpus granatum and the other comprising a mangrove Xylocarpus moluccensis and a terrestrial Xylocarpus rumphii. In conjunction with previous studies, we identified several genomic features associated with mangroves: (i) signals of positive selection in genes related to salt tolerance and root development; (ii) genome-wide elevated ratios of non-synonymous to synonymous substitution relative to terrestrial relatives; and (iii) active elimination of long terminal repeats. These features are found in the terrestrial X. rumphii in addition to the two mangroves. These genomic features, not being strictly mangrove-specific, are hence considered pre-adaptive. We infer that the coastal but non-intertidal habitat of X. rumphii may have predisposed the common ancestor to invasion of true mangrove habitats. Other features including the preferential retention of duplicated genes and intolerance to pseudogenization are not found in X. rumphii and are likely true adaptive features in mangroves. In conclusion, by studying adaptive shift and partial shifts among closely related species, we set up a framework to study genomic features that are acquired at different stages of the pre-adaptation and adaptation to new environments.

Where whole-genome duplication is most beneficial: Adaptation of mangroves to a wide salinity range between land and sea.

Whole-genome duplication (WGD) is believed to increase the chance of adaptation to a new environment. This conjecture may apply particularly well to new environments that are not only different but also more variable than ancestral habitats. One such prominent environment is the interface between land and sea, which has been invaded by woody plants, collectively referred as mangroves, multiple times. Here, we use two distantly related mangrove species (Avicennia marina and Rhizophora apiculata) to explore the effects of WGD on the adaptive process. We found that a high proportion of duplicated genes retained after WGD have acquired derived differential expression in response to salt gradient treatment. The WGD duplicates differentially expressed in at least one copy usually (>90%) diverge from their paralogues' expression profiles. Furthermore, both species evolved in parallel to have one paralogue expressed at a high level in both fresh water and hypersaline conditions but at a lower level at medium salinity. The pattern contrasts with the conventional view of monotone increase/decrease as salinity increases. Differentially expressed copies have thus probably acquired a new role in salinity tolerance. Our results indicate that the WGD duplicates may have evolved to function collaboratively in coping with different salinity levels, rather than specializing in the intermediate salinity optimal for mangrove plants. In conclusion, WGD and the retained duplicates appear to be an effective solution for adaptation to new and unstable environments.

Evolution of woody plants to the land-sea interface - The atypical genomic features of mangroves with atypical phenotypic adaptation.

How plants adapt and diverge in extreme environments is a key question of plant evolution and ecology. Mangrove invasion of intertidal environments is facilitated by adaptive phenotypes such as aerial roots, salt-secreting leaf, and viviparity, and genomic mechanisms including whole genome duplication and transposable element number reduction. However, a number of mangroves lack these typical phenotypes. The question we ask is whether these phenotypically atypical mangroves also have distinct genomic features? The sibling mangrove species Lumnitzera littorea and Lumnitzera racemosa provide a model to study this question. We sequenced and assembled their genomes to chromosome level, together with a closely related species Combretum micranthum. While most mangroves have small genomes, the genomes of both Lumnitzera species are large (1443 and 1317 Mb) and carry a high proportion of repeat sequences (~75%). Moreover, Lumnitzera species have not undergone post-gamma whole-genome duplications. Their genome size increased mainly due to the expansion of repeat sequences in their ancestors. However, Lumnitzera genomes have reduced transposable elements by constraining the proliferation of new LTR-RTs. Meanwhile, the two species have more gene families contracted than expanded, and some gene families with reversed size change may underlie their differentiation in root morphology and local distribution. We identified 86 chromosomal inversions, five of which are measured between 6.5 and 12.8 megabases. A number of genes located in these inversions function in pigment biosynthesis, a process likely involved in flower colour differentiation between the Lumnitzera species. We conclude that the mangroves with atypical phenotypes also have atypical genomic evolution.

Interplay of Drop Shedding Mechanisms on High Wettability Contrast Biphilic Stripe-Patterned Surfaces.

To improve the rate of DWC, numerous studies have adjusted the distribution of drops through biphilic surface patterning and wettability gradients to control the nucleation and drop shedding rates on the condensing surface, yet the connection between drop shedding mechanisms and surface wettability patterning remains unclear. Moreover, wettability patterning places geometric bounds on the governing forces (i.e., gravity, capillary, and inertia), which drive the droplet shedding mechanisms. Thus, the subsequent influence of droplet distribution along the DWC regions on the shedding mechanisms may not be known a priori. In this study, the area fraction, ADWC, of the DWC and also the DWC region width, LN, were varied between 10 and 50% and 0.5-1.5 mm, respectively, to probe the dominant droplet shedding mechanisms on a high wettability contrast surface (i.e., the contact angle on the DWC was 159 ± 3.4° and the hysteresis 9 ± 3.6°, whereas the FWC was nearly perfectly wetting). Humid air was introduced inside a custom-built chamber with the upright steady-state condensation imaged by both real-time and high-speed imaging techniques. We found that the droplet shedding mechanisms changed with increasing LN where the sliding drop radii are reduced with LN while the jumping drop radii remained unchanged with LN. The maximum drop size for shedding also decreased by 13%, which we attribute to the secondary droplet inertia, which helps gravity overcome the capillary retention force. Lastly, although many studies have probed DWC enhancements via surface wettability patterning, an optimal combination of ADWC and LN provided in this study significantly aids in the improvement of future DWC-based condensers and water collector applications.

Chromosomal-Scale Genome Assemblies of Two Coastal Plant Species, Scaevola taccada and S. hainanensis-Insight into Adaptation Outside of the Common Range.

While most of the species in Goodeniaceae family, excluding the Scaevola genus, are endemic to Australasia, S. taccada and S. hainanensis have expanded their distribution range to the tropical coastlines of the Atlantic and Indian Oceans. S. taccada appears to be highly adapted to coastal sandy lands and cliffs, and it has become invasive in places. S. hainanensis is found mainly in salt marshes near mangrove forests, and is at risk of extinction. These two species provide a good system to investigate adaptive evolution outside the common distribution range of this taxonomic group. Here, we report their chromosomal-scale genome assemblies with the objective of probing their genomic mechanisms related to divergent adaptation after leaving Australasia. The scaffolds were assembled into eight chromosome-scale pseudomolecules, which covered 90.12% and 89.46% of the whole genome assembly for S. taccada and S. hainanensis, respectively. Interestingly, unlike many mangroves, neither species has undergone whole-genome duplication. We show that private genes, specifically copy-number expanded genes are essential for stress response, photosynthesis, and carbon fixation. The gene families that are expanded in S. hainanensis and contracted in S. taccada might have facilitated adaptation to high salinity in S. hainanensis. Moreover, the genes under positive selection in S. hainanensis have contributed to its response to stress and its tolerance of flooding and anoxic environments. In contrast, compared with S. hainanensis, the more drastic copy number expansion of FAR1 genes in S. taccada might have facilitated its adaptation to the stronger light radiation present in sandy coastal lands. In conclusion, our study of the chromosomal-scale genomes of S. taccada and S. hainanensis provides novel insights into their genomic evolution after leaving Australasia.

Oscillation Dynamics of Drops on Immiscible Thin Liquid Films.

While drop oscillation dynamics has been widely studied for many decades, the influence of a moving contact line on the oscillation modes of drops remains underexplored. Herein, we report the oscillation dynamics of drops on thin liquid films with different viscosities where lower viscosities provide a slipping surface and higher viscosities immobilize the contact line. A gently deposited drop onto an oil film undergoes shape oscillations due to capillarity, where the frequency, amplitude, and apparent contact angle are tracked via a high-speed camera. This study demonstrates that restraining the mobility of the drop contact line by increasing the viscosity of a thin oil film underneath the drop increases the extent of the drop oscillation time as well as affecting the natural frequency of the drop oscillation. The drop oscillation time was defined by the time at which the changes in the drop height dropped to values less than 1% of the equilibrium height. The experimental results for the first longitudinal mode oscillation frequencies as a function of the equilibrium contact angles for the pinning and slipping contact lines were in good agreement with previously reported numerical simulations and model predictions.

Microwave frequency measurement using a silicon integrated microring resonator.

Photonics-assisted instantaneous frequency measurement of a microwave signal using a silicon integrated microring resonator (MRR) is proposed and experimentally demonstrated. The frequency of a microwave signal has a unique relationship with the power ratio between the two microwave signals at the outputs of two microwave photonic filters (MPF) with complementary frequency responses. The key device to implement the MPFs is a silicon integrated MMR, which is employed to convert a phase-modulated optical signal to an intensity-modulated optical signal by placing two optical carriers at the complementary slopes of the MRR. For a given frequency measurement range and resolution, an MRR is designed and fabricated, and its use for instantaneous microwave frequency (IMF) measurement is implemented. For the fabricated MRR, an IMF measurement range of 14-25 GHz with a measurement accuracy of ±0.2GHz is achieved.

Genomic insights into molecular adaptation to intertidal environments in the mangrove Aegiceras corniculatum.

Mangroves have colonised extreme intertidal environments characterised by high salinity, hypoxia and other abiotic stresses. Aegiceras corniculatum, a pioneer mangrove species that has evolved two specialised adaptive traits (salt secretion and crypto-vivipary) is an attractive ecological model to investigate molecular mechanisms underlying adaptation to intertidal environments. We assembled de novo a high-quality reference genome of A. corniculatum and performed comparative genomic and transcriptomic analyses to investigate molecular mechanisms underlying adaptation to intertidal environments. We provide evidence that A. corniculatum experienced a whole-genome duplication (WGD) event c. 35 Ma. We infer that maintenance of cellular environmental homeostasis is an important adaptive process in A. corniculatum. The 14-3-3 and H+ -ATPase protein-coding genes, essential for the salt homeostasis, were preferentially retained after the recent WGD event. Using comparative transcriptomics, we show that genes upregulated under high-salt conditions are involved in salt transport and ROS scavenging. We also found that all homologues of DELAY OF GERMINATION1 (DOG1) had lost their heme-binding ability in A. corniculatum, and that this may contribute to crypto-vivipary. Our study provides insight into the genomic correlates of phenotypic adaptation to intertidal environments. This could contribute not only within the genomics community, but also to the field of plant evolution.

Low jitter microwave pulse train generation based on an optoelectronic oscillator.

We demonstrate an approach to ultra-short pulse train generation with a low time jitter based on pulse compression of a frequency comb generated by a dual-loop optoelectronic oscillator (OEO). The proposed dual-loop OEO consists of two feedback loops, with one having a long loop length and the other a short loop length. In the long loop, a phase modulator (PM) cascaded with a Mach-Zehnder modulator (MZM) are employed, and in the short loop, only the MZM is included. Due to the Vernier effect, the use of the dual-loop structure can facilitate mode selection to generate a single-frequency microwave carrier with multiple optical sidebands corresponding to an optical comb. By adjusting the phase relationship between the optical sidebands using a dispersion compensating fiber (DCF), a stable optical pulse train is generated. Thanks to the low phase noise nature of an OEO, the generated pulse train has a low time jitter. The proposed approach is evaluated experimentally. A pulse train with a repetition frequency of 2.023 GHz and a pulse width of 40 ps is generated. The single-sideband (SSB) phase noise of the carrier frequency generated by the OEO is measured to be -118 dBc/Hz at a 10-kHz offset frequency, corresponding to a time jitter of the pulse train of 391.2 fs. The phase noise can be further reduced if an active cavity stabilization mechanism is adopted, enabling further reduction in the time jitter to the order of tens of femtoseconds.

Drop Bouncing Dynamics on Ultrathin Films.

For drops to contact various surfaces, the removal of the interstitial fluid is the prerequisite to contact. While the conventional understanding is for drops to irreversibly spread on a film made of the same substance, we describe the dynamics of drops initiating contact yet carrying enough momentum to completely lift off of the substrate which we label as contact bouncing. We report new experimental results of the dynamics between drops impacting thin films described by the ratio of the liquid film hL to the drop with diameter D0 for the range of 0.004 < hL/D0 < 0.08. Using high-speed interferometry, we visualize the interfacial gas layer spatiotemporal signatures across the various film thicknesses and Weber numbers. We find that while increasing the deformability of the thin films enhances the gas entrainment phenomenon at early times, it also increases the rate of the gas purging rate, increasing the chance of contact just prior to the gas film retraction and drop lift off sequence. Drops which contact the liquid film during the retraction stage are able to bounce with <5% volume loss.

Molecular Evolution in Large Steps-Codon Substitutions under Positive Selection.

Molecular evolution is believed to proceed in small steps. The step size can be defined by a distance reflecting physico-chemical disparities between amino acid (AA) pairs that can be exchanged by single 1-bp mutations. We show that AA substitution rates are strongly and negatively correlated with this distance but only when positive selection is relatively weak. We use the McDonald and Kreitman test to separate the influences of positive and negative selection. While negative selection is indeed stronger on AA substitutions generating larger changes in chemical properties of AAs, positive selection operates by different rules. For 65 of the 75 possible pairs, positive selection is comparable in strength regardless of AA distance. However, the ten pairs under the strongest positive selection all exhibit large leaps in chemical properties. Five of the ten pairs are shared between Drosophila and Hominoids, thus hinting at a common but modest biochemical basis of adaptation across taxa. The hypothesis that adaptive changes often take large functional steps will need to be extensively tested. If validated, molecular models will need to better integrate positive and negative selection in the search for adaptive signal.

Molecular adaptation to salinity fluctuation in tropical intertidal environments of a mangrove tree Sonneratia alba.

BACKGROUND: Mangroves have adapted to intertidal zones - the interface between terrestrial and marine ecosystems. Various studies have shown adaptive evolution in mangroves at physiological, ecological, and genomic levels. However, these studies paid little attention to gene regulation of salt adaptation by transcriptome profiles. RESULTS: We sequenced the transcriptomes of Sonneratia alba under low (fresh water), medium (half the seawater salinity), and high salt (seawater salinity) conditions and investigated the underlying transcriptional regulation of salt adaptation. In leaf tissue, 64% potential salinity-related genes were not differentially expressed when salinity increased from freshwater to medium levels, but became up- or down-regulated when salt concentrations further increased to levels found in sea water, indicating that these genes are well adapted to the medium saline condition. We inferred that both maintenance and regulation of cellular environmental homeostasis are important adaptive processes in S. alba. i) The sulfur metabolism as well as flavone and flavonol biosynthesis KEGG pathways were significantly enriched among up-regulated genes in leaves. They are both involved in scavenging ROS or synthesis and accumulation of osmosis-related metabolites in plants. ii) There was a significantly increased percentage of transcription factor-encoding genes among up-regulated transcripts. High expressions of salt tolerance-related TF families were found under high salt conditions. iii) Some genes up-regulated in response to salt treatment showed signs of adaptive evolution at the amino acid level and might contribute to adaptation to fluctuating intertidal environments. CONCLUSIONS: This study first elucidates the mechanism of high-salt adaptation in mangroves at the whole-transcriptome level by salt gradient experimental treatments. It reveals that several candidate genes (including salt-related genes, TF-encoding genes, and PSGs) and major pathways are involved in adaptation to high-salt environments. Our study also provides a valuable resource for future investigation of adaptive evolution in extreme environments.

Genome-Wide Convergence during Evolution of Mangroves from Woody Plants.

When living organisms independently invade a new environment, the evolution of similar phenotypic traits is often observed. An interesting but contentious issue is whether the underlying molecular biology also converges in the new habitat. Independent invasions of tropical intertidal zones by woody plants, collectively referred to as mangrove trees, represent some dramatic examples. The high salinity, hypoxia, and other stressors in the new habitat might have affected both genomic features and protein structures. Here, we developed a new method for detecting convergence at conservative Sites (CCS) and applied it to the genomic sequences of mangroves. In simulations, the CCS method drastically reduces random convergence at rapidly evolving sites as well as falsely inferred convergence caused by the misinferences of the ancestral character. In mangrove genomes, we estimated ∼400 genes that have experienced convergence over the background level of convergence in the nonmangrove relatives. The convergent genes are enriched in pathways related to stress response and embryo development, which could be important for mangroves' adaptation to the new habitat.

Convergent adaptive evolution in marginal environments: unloading transposable elements as a common strategy among mangrove genomes.

Several clades of mangrove trees independently invade the interface between land and sea at the margin of woody plant distribution. As phenotypic convergence among mangroves is common, the possibility of convergent adaptation in their genomes is quite intriguing. To study this molecular convergence, we sequenced multiple mangrove genomes. In this study, we focused on the evolution of transposable elements (TEs) in relation to the genome size evolution. TEs, generally considered genomic parasites, are the most common components of woody plant genomes. Analyzing the long terminal repeat-retrotransposon (LTR-RT) type of TE, we estimated their death rates by counting solo-LTRs and truncated elements. We found that all lineages of mangroves massively and convergently reduce TE loads in comparison to their nonmangrove relatives; as a consequence, genome size reduction happens independently in all six mangrove lineages; TE load reduction in mangroves can be attributed to the paucity of young elements; the rarity of young LTR-RTs is a consequence of fewer births rather than access death. In conclusion, mangrove genomes employ a convergent strategy of TE load reduction by suppressing element origination in their independent adaptation to a new environment.

Extremely low genetic diversity across mangrove taxa reflects past sea level changes and hints at poor future responses.

The projected increases in sea levels are expected to affect coastal ecosystems. Tropical communities, anchored by mangrove trees and having experienced frequent past sea level changes, appear to be vibrant at present. However, any optimism about the resilience of these ecosystems is premature because the impact of past climate events may not be reflected in the current abundance. To assess the impact of historical sea level changes, we conducted an extensive genetic diversity survey on the Indo-Malayan coast, a hotspot with a large global mangrove distribution. A survey of 26 populations in six species reveals extremely low genome-wide nucleotide diversity and hence very small effective population sizes (Ne ) in all populations. Whole-genome sequencing of three mangrove species further shows the decline in Ne to be strongly associated with the speed of past changes in sea level. We also used a recent series of flooding events in Yalong Bay, southern China, to test the robustness of mangroves to sea level changes in relation to their genetic diversity. The events resulted in the death of half of the mangrove trees in this area. Significantly, less genetically diverse mangrove species suffered much greater destruction. The dieback was accompanied by a drastic reduction in local invertebrate biodiversity. We thus predict that tropical coastal communities will be seriously endangered as the global sea level rises. Well-planned coastal development near mangrove forests will be essential to avert this crisis.

Can genomic data alone tell us whether speciation happened with gene flow?

The allopatric model, which requires a period of geographical isolation for speciation to complete, has been the standard model in the modern era. Recently, "speciation with gene flow" has been widely discussed in relation to the model of "strict allopatry" and the level of DNA divergence across genomic regions. We wish to caution that genomic data by themselves may only permit the rejection of the simplest form of allopatry. Even a slightly more complex and realistic model that starts with subdivided populations would be impossible to reject by the genomic data alone. To resolve this central issue of speciation, other forms of observations such as the sequencing of reproductive isolation genes or the identification of geographical barrier(s) will be necessary.