Introduction

Plants are adapted to a wide range of environments over the earth and show specific adaptations to extreme environments, such as aquatic biospheres, high mountains, deserts, special soils, and so forth (Baker et al. 1992; Hoffman and Parsons 1997; Warton 2002; Körner 2003; Nagy and Grabherr 2009; Ramawat 2010; Evert and Eichhorn 2013). Among such plants, Podostemaceae are extreme aquatic haptophytes growing on waterworn rock surfaces in fast turbulent currents in rapids and waterfalls (Willis 1914; Sculthope 1967; Kato 2013). The currents sweeping the rock surfaces exclude macrophytes. Furthermore, Podostemaceae are restricted to seasonal fluctuations of water level in which the plants are exposed and flower above the lowered water level in the dry season. Such habitats are also inhabited by Hydrostachyaceae in some localities in Madagascar and Africa (Cusset 1972, 1973). Because Hydrostachyaceae do not occur outside those regions, they are not competitors of Podostemaceae, such as Hydrobryum, in Asia.

Although all habitats are in running water above stable rocks or solid substrates, the morphology of Podostemaceae is unique and diverse. In sharp contrast to the anchoring, absorbent subterranean roots of terrestrial plants, their root is a major adhering organ, while the stem, leaves and flowers are adventitious from the roots and, in Asian species of the subfamily Podostemoideae, the stem is usually reduced. Even in the early life history during early rainy season, the root, together with adhesive hairs on the hypocotyl, adheres to the rock surface. Some Podostemaceae do not have even a set of fundamental organs. A few genera are rootless (Imaichi et al. 2004; Cook and Rutishauser 2007; Koi and Kato 2010), and the stems of many genera are atypical, lacking a shoot apical meristem (Imaichi et al. 2005; Koi et al. 2005). Unlike most angiosperms and the Podostemaceae subfamilies Tristichoideae and Weddellinoideae, members of the Podostemoideae show borderless or fuzzy shoot-leaf morphology (Rutishauser 1995), which is consistent with the unique pattern of gene expression (Katayama et al. 2010).

The epilithic roots are flattened to various degrees and dorsiventral, playing the role of adhesion to rock surfaces, adaptation to fast currents and photosynthesis (like leaves), which are essential for the haptophytic life. The root form ranges from subcylindrical and ribbon-like to foliose or disk-like (Koi et al. 2006; Cook and Rutishauser 2007). In Asian Podostemoideae, the ribbon-like roots are characterized by their constant width, a single strand of elongate nontracheary cells running into the root apex and a root-borne shoot occurring at a sinus of root branches, whereas the foliose roots are characterized by their spreading width (fan shape), a network of strands of elongate nontracheary cells and root-borne shoots scattered on the dorsal surface of the root independently of root branching (Ota et al. 2001; Hiyama et al. 2002; Koi and Kato 2003; Koi et al. 2006). There are foliose roots (Fig. 1a) in, e.g., Hanseniella, Hydrobryum, Thawatchaia and Zeylanidium in Asia (Hiyama et al. 2002; Kato 2004; Koi and Kato 2012), while ribbon-like roots (Fig. 1b) are in, e.g., Cladopus, Paracladopus and Zeylanidium (Hiyama et al. 2002; Koi and Kato 2003, 2012; Kato 2006; Koi et al. 2008; Kato and Koi 2018). The root forms are produced by the root meristem, which is also dorsiventral to varying degrees (Koi et al. 2006). Koi et al. (2006) found that the root is primitive in the subfamilies Tristichoideae and Weddellinoideae, while the root of the derived Podostemoideae is specialized, being foliose with marginal meristems (Ota et al. 2001).

Fig. 1
figure 1

The illustrations are reproduced from Koi and Kato (2012)

Morphology of Podostemaceae roots. a Foliose root (Hydrobryum subcrustaceum). b Ribbon-like root (H. taeniatum). F flower; R root. Scale bars = 5 mm (a) and 3 mm (b).

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The aquatic Podostemaceae are sister to the phanerophytic Hypericaceae (Ruhfel et al. 2011; Xi et al. 2012). After the earliest adaptation during this drastic habitat change, the subsequent evolution and adaptation of root forms and other characters always took place in riverine habitats, where all species (> 300 spp.) evolved. It may be possible that diversification occurred in competitor-free habitats with relaxed natural selection. Based on his worldwide knowledge of the ecology, morphology and distribution, Willis (1914) argued that the great variation in Podostemaceae was produced in the absence of adaptation. This idea was taken over by van Steenis (1969, 1977, 1978, 1981). However, such evolution has not been demonstrated based on specific data.

In a phylogenetic analysis of the root form, Koi et al. (2006) and Moline et al. (2007) stressed that the foliose roots of Hydrobryum and a few allied genera are derived from the ribbon-like roots of, e.g., Cladopus. Hydrobryum is a most specialized genus of Asian Podostemoideae. Several species of Hydrobryum examined consist of a hypocotyl and two cotyledons but are devoid of a primary shoot and root in the embryo-seedling, and the root is adventitious and exogenous, instead of endogenous as in primitive genera and most other families (Suzuki et al. 2002; Koi et al. 2012b). The roots of 29 of the 33 species of Hydrobryum, as in allied genera, are foliose with leaves and shoots scattered irregularly on the dorsal surface (Fig. 1a) (Ota et al. 2001; Koi and Kato 2012, 2015b, 2018, 2019; Kato 2013, 2018). The roots of the remaining four species are ribbon-like with tufted leaves and flowers borne regularly in the sinuses of root branches (Fig. 1b) (Koi and Kato 2012, 2018), quite similar to the roots of Cladopus. Although molecular phylogenetic data indicate that the ribbon-like species of Hydrobryum are related to foliose species, their evolutionary processes have not been discussed (Koi and Kato 2012, 2019). In spite of the different root forms, the species are sympatric. However, distribution information is incomplete and requires further field data to determine the microdistribution.

The aim of this study was to examine whether the evolution of the root form in Hydrobryum is adaptive, by analyzing its phylogeny and comparing it with the microdistribution of the species.

Materials and methods

Materials and collection sites

Hydrobryum hapteron, H. ramosum, H. stellatum, H. subcrustaceum, H. subcylindricoides, H. subcylindricum, H. taeniatum, H. takakioides and/or H. verrucosum occur in ten waterfalls and rapids in one mountain range northeast of Vientiane, Laos. Six waterfalls and rapids flow into one tributary of the Mekong River. Most sites are in the Phou Khao Khouay National Protected Area (NPA), and the Tad Nampa waterfall is near the eastern margin of the Area. Materials collected were dried with silica gel for molecular phylogenetic analysis (Online Resource 1) or fixed with FAA (formalin/acetic acid/50% ethyl alcohol = 5:5:90 in volume) for morphological observations (Table 1). Vouchers are deposited in the Department of Botany, National Museum of Nature and Science (TNS) and the National Herbarium of Laos (HNL).

Table 1 Materials used in morphological observations
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The localities of the materials are generally from east to west. The easternmost and westernmost waterfalls are nearly 100 km apart, while Tad Leuk and the rapids upstream of Tad Xai are about 3 km apart from Tad Yong and Tad Xai, respectively (Fig. 2). Each waterfall is inhabited by two or more, rarely one species of Hydrobryum. Species of Dalzellia, Hydrodiscus and Polypleurum also coexist with the species of Hydrobryum (Koi and Kato 2012, 2015a, b). In other waterfalls within the NPA, there is Hydrobryum vientianense, which belongs to another clade (Koi and Kato 2012). Species that are not monophyletic with H. austrolaoticum (see Results) were excluded from analysis. The species of Hydrobryum analyzed, except H. verrucosum, were not found outside the Area during 13 recent explorations in Laos (Koi and Kato 2012, 2015a, b, 2018, 2019; Koi et al. 2019) (Table 1).

Fig. 2
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Map showing localities (rapids or waterfalls) of species of Hydrobryum in Phou Khao Khouay NPA. Inset shows location in Laos. 1, Chaova; 2, Nam Mang 3 Dam; 3, Tad Yong; 4, Tad Leuk; 5, Tad Xai; 6, waterfall in Xai stream, upstream of Tad Xai; 7, rapids upstream of Tad Xai; 8, Tad Lo; 9, Tad Heua Hak; and 10, Tad Nampa. The numbers correspond with those in Table 2

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Molecular phylogeny

Chloroplast matK gene sequences (1524 bp) were determined using the methods described previously by Koi and Kato (2010). Sequences deposited in GenBank were used for phylogenetic analyses (Online Resource 1). The sequences were aligned by Clustal X ver. 2.1 (Larkin et al. 2007) and refined manually with Mesquite ver. 3.40 (Maddison and Maddison 2017) (Online Resource 2). Gaps were treated as missing data. The program MrModeltest 2.3 (Nylander 2004) determined a general time reversible (GTR) + proportion of invariable sites (I) + shape parameter of the gamma distribution (G) substitution model as the best fitting model of substitution: Nucleotide frequencies were A = 0.3342, C = 0.1385, G = 0.1231, T = 0.4042; the substitution rate matrix was A to C = 1.1948, A to G = 1.0928, A to T = 0.2083, C to G = 0.4480, C to T = 0.8737, G to T = 1.0000; and the proportion of invariable sites was 0.2425. The gamma distribution shape parameter was 0.8650. Maximum likelihood (ML) analysis was conducted using RAxML-HPC2 (Stamatakis 2014) on XSEDE (8.2.10) in Cipres Science Gateway (Miller et al. 2010) with GTR + I + G model. Bootstrap probability (BP) values were calculated for 1000 replicates. In maximum parsimony (MP) analyses with the program PAUP* Version 4.0a159 (Swofford 2002), all characters were equally weighted, and bootstrap values were calculated for 10,000 replicates with ten random addition replicates involving tree-bisection-reconnection (TBR) branch swapping; the ‘MulTrees’ option was not in effect. Endocaulos mangorense and Thelethylax minutiflora were used as outgroups (Koi et al. 2012a).

Morphology

For morphological studies, we used specimens preserved in FAA. The roots were observed under a stereo microscope (Leica M125, Heerbrugg, Switzerland), focusing on the site where shoots are formed. Observations considered the following developmental features of the roots and shoots: In Cladopus allied to Hydrobryum, the shoots are initiated within the root meristem and the root is divided into two unequal branches with shoots borne in the sinuses of the root branches (Koi and Kato 2003). A quite similar mode of shoot development is reported for Zeylanidium, which is phylogenetically far from the Cladopus–Hydrobryum clade (Hiyama et al. 2002). In comparison, in the foliose roots of Hydrobryum and Zeylanidium, the shoots are formed proximally to the root meristem and are not involved in the division of the root, so that the shoots are scattered on the surface of the root (Fig. 1a; Ota et al. 2001; Hiyama et al. 2002).

Character phylogeny

The evolution of the root form was reconstructed onto the present phylogenetic tree of the species using the program Mesquite ver. 3.31 (Maddison and Maddison 2017). The character states, foliose and ribbon-like, were derived from our previous studies (Koi and Kato 2012, 2015b, 2018, 2019; Kato 2013, 2018; Koi et al. 2019).

Results

Phylogeny

Hydrobryum was divided into three clades, i.e., a clade of H. bifoliatum/H. kaengsophense/H. phurueanum, a clade of H. chompuense/H. mandaegense/H. tardhuangense/H. varium and the remaining species (Fig. 3a). The former two clades were supported with 100% bootstrap values in both RAxML and MP analyses, while the latter was supported with 70% and 74% bootstrap values in RAxML and MP analyses, respectively. The last clade was subdivided into five subclades with robust support. One of the subclades was further divided into H. austrolaoticum and the rest. In the last subclade, the three species with ribbon-like roots were separately sister to the three species with foliose roots: The group (Fig. 3a, Clade i-b) of H. taeniatum and H. subcylindricum was sister to a group (Clade i-a) of H. takakioides and H. stellatum (Clade i); Hydrobryum subcylindricoides was sister to H. subcrustaceum (Clade ii); and the group (Clade iii-a) of H. ramosum and H. hapteron was sister to the group (Clade iii-b) of H. verrucosum and H. clandestinum.

Fig. 3
figure 3figure 3

Phylogenetic relationships of aHydrobryum and allies (i.e., Cladopus–Hydrobryum clade), and bZeylanidium and allies (i.e., Polypleurum–Zeylanidium clade) inferred from RAxML analysis based on matK sequence. Monophyletic species of podostemoid genera related to Hydrobryum are represented by triangles (with vertical width reflecting the number of samples). Support values (≥ 50) of RAxML and MP are shown above and below branches. Asterisks indicate branches supported with 100% bootstrap values of RAxML and MP analyses

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Root morphology and character phylogeny

The roots of H. clandestinum, H. hapteron, Hydrobryum stellatum and H. subcrustaceum, as well as H. takakioides and H. verrucosum (data not shown), are foliose with irregular lobes (Figs. 1a, 4a, c, e, g). The vegetative shoots consisting of tufted leaves and the floriferous shoots are scattered on the dorsal surface of the roots with no or little relationship to the division of the roots. The roots of H. taeniatum, H. ramosum, H. subcylindricum and H. subcylindricoides are ribbon-like (Figs. 1b, 4b, d, f, h). They are sympodially or dichotomously branched with the vegetative shoots or floriferous shoots in the sinuses of root branches. This morphology is quite similar to that of Cladopus and other related genera in Asian podostemoids. We observed young shoots in the root meristematic areas in H. taeniatum (Fig. 4b) and H. ramosum (data not shown), like in Zeylanidium lichenoides (Hiyama et al. 2002), although histological data are not available for shoot development.

Fig. 4
figure 4

Roots of Hydrobryum species. a Foliose root of Hydrobryum clandestinum with leaves scattered on dorsal surface. b Ribbon-like root of H. taeniatum with leaves in sinuses of root branches. Note shoot primordium in green color appeared at root apex. c–h Foliose (c, e, g) and ribbon-like (d, f, h) roots of sister species of Hydrobryum. cHydrobryum hapteron, dH. ramosum, eH. stellatum, fH. subcylindricum, gH. subcrustaceum, hH. subcylindricoides. Asterisks indicate root branches. Arrows indicate tufted leaves (a, b), flower buds (c, d, e, h) or fruits (f, g). Scale bars = 5 mm

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The character phylogeny of root form showed that the foliose root is an ancestral state in Hydrobryum, while the ribbon-like root is a derived state (Fig. 5). The ribbon-like roots were derived three times from the foliose roots in the clade of H. stellatum, H. takakioides, H. taeniatum and H. subcylindricum, in the clade of H. subcrustaceum and H. subcylindricoides and in the clade of H. hapteron and H. ramosum (Fig. 5, arrows). In contrast, the ribbon-like root is ancestral in the common ancestor of the Cladopus and Hydrobryum clades (Fig. 5, arrowhead). In the Polypleurum–Zeylanidium clade, the foliose root is derived recurrently in Griffithella hookeriana, Willisia, Z. crustaceum and Z. olivaceum/Z. maheshwarii (Figs. 3b, 5).

Fig. 5
figure 5

Phylogenetic relationship of root form in Asian Podostemoideae. Arrowhead indicates the common ancestor of the Cladopus–Hydrobryum clade. Arrows indicate the branching points where root form evolved from foliose to ribbon-like

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Distribution

In total, 15 species were found in ten waterfalls and rapids in the Phou Khao Khouay NPA (Table 2, Fig. 2). Eight of the localities (Loc. 2–8, 10) are inhabited by sympatric species. Five foliose or ribbon-like species, i.e., H. taeniatum, H. subcylindricum, H. subcrustaceum, H. verrucosum and H. clandestinum, each occur in two or more localities, while the other five, H. takakioides, H. stellatum, H. subcylindricoides, H. hapteron and H. ramosum, are restricted to single localities. The foliose and ribbon-like species are sympatric in six localities (Loc. 2–5, 7, 10) and occasionally occur even on the same rock.

Table 2 Microdistribution of sister species of Hydrobryum and species of other genera in waterfalls or rapids in Phou Khao Khouay NPA. Clades correspond with those in Fig. 3
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Discussion

Two-way evolution

The study of character evolution suggests that the foliose root recently changed three times to the ribbon-like root in Hydrobryum. In comparison, the foliose roots had earlier been derived from the ribbon-like roots of the common ancestor of Cladopus, Paracladopus, Ctenobryum, Hydrodiscus, Hanseniella, Thawatchaia and Hydrobryum and was derived recurrently in Griffithella hookeriana, Willisia (2 spp.) and Zeylanidium pro parte (present study; Koi et al. 2006; Moline et al. 2007). The ribbon-like roots of Hydrobryum exactly share the root–shoot spatial relationship with those of Cladopus, Zeylanidium and several species of Polypleurum (Mathew and Satheesh 1997; Kato 2006). Taking into account the specific mode of root–shoot development, as noted in Introduction and Methods (Ota et al. 2001; Hiyama et al. 2002; Koi and Kato 2003), and the recurrent sister–species relationships of root forms, the derived and the ancestral ribbon-like roots are comparable with each other. Therefore, we estimate that evolution of the root form is two-way and recurrent in the Cladopus–Hydrobryum clade. It is plausible that the derived ribbon-like root is produced by a slight genetical change in the process of development, e.g., a reuse of the pathway of the ancestral root, although comparative developmental and molecular genetic studies are necessary to reveal the evolutionary mechanism.

Sympatry

The foliose species (F) and ribbon-like species (R) of Hydrobryum are sympatric in six of ten localities in the area (Loc. 2–5, 7, 10 in Table 2). The two types of roots are semicircular in outline because root branching occurs near the apex of the ribbon roots, and the species of the two types usually occur on different rocks or separately on the same wide rocks. Among them, two species in Nam Mang 3 Dam (Loc. 2) are sister to those in rapids upstream of Tad Xai (Loc. 7): H. subcrustaceum (F) to H. subcylindricoides (R) (Clade ii) and H. ramosum (R) to H. hapteron (F) (Clade iii-a). Most likely, they are products of allopatric speciation. [Allopatric speciation is also suggested for H. takakioides (F) and H. stellatum (F) (Clade i-a), and less clearly for H. verrucosum (F) and H. clandestinum (F) (Clade iii-b), although the sister species retain the same root form.] These allopatric species are sympatric with species of different phylogenies. The sympatry in Loc. 2 and Loc. 7 perhaps appeared at the time of speciation. By contrast, the ribbon-like H. taeniatum and H. subcylindricum (Clade i-b) diverged from the common ancestor with foliose H. takakioides and H. stellatum (Clade i-a), indicating a longer history of sympatry in Loc. 3 and Loc. 5. Sympatry in the other Loc. 4 and Loc. 10 is shown by ribbon-like and foliose species with other patterns of phylogeny. Such frequent and recurrent sympatry strongly suggests that the two root forms have equivalent adhesion capability of roots, which is essential for the haptophytic life, although other differences are not excluded.

In other areas, too, foliose species of Hydrobryum are sympatric with ribbon-like species of Cladopus in, e.g., two waterfalls in Khao Yai National Park, central Thailand; a waterfall in Sa Kaeo, eastern Thailand; and two rapids in Kagoshima, Japan (Kato 2004, 2006, 2008; Koi and Kato 2012; Werukamkul et al. 2018; Kato unpubl. observations). Furthermore, species of other genera with different root forms or holdfasts coexist in the same localities worldwide (Table 2; Willis 1914; Philcox 1996; Mathew and Satheesh 1997; Novelo and Philbrick 1997; Rutishauser 1997; Tur 1997; Kato 2004, 2006; Lin et al. 2016; Werukamkul et al. 2012; Kato and Koi unpubl. observations). Sympatry in a wide range of species and areas strengthens the suggested equivalent capability of the different root forms.

Nonadaptive evolution

Contrary to neutral evolution at the molecular level (Kimura 1983), phenotypic evolution is generally accepted as being adaptive, but Lewontin (1978) using the number of horns of rhinoceros and other examples, Nei (1987, 2013), Stearns and Hoekstra (2005) and Saitou (2009) stressed that it may not always be so. Darwin (1859) also mentioned that natural selection has been the main but not the exclusive means of modification. From the viewpoint of adaptive evolution (e.g., Bell 2008; Futuyma 2009), theoretically, in the early evolution, the derived foliose root of the Hydrobryum clade would have gained larger advantages than the ancestral ribbon-like root. Given that this trend continued in the late evolution, as seen in the Polypleurum–Zeylanidium clade (this study), the derived ribbon-like root in Hydrobryum should have received disadvantages. This interpretation seems unlikely, and instead we interpret the two-way root evolution as nonadaptive or nearly so and not as adaptive convergence (Rutishauser and Moline 2005). It is consistent with our findings that the foliose and ribbon-like species are sympatric, irrespective of phylogenetic relationships. Evolution of Podostemaceae appears to have occurred in the specific habitats that are monopolized by Podostemaceae and have little natural selection with competing macrophytes. This study may provide the first empirical data consistent with the idea of nonadaptive evolution of phenotypic characters in Podostemaceae (Willis 1914; see Introduction). However, data of microdistribution of sister species in the natural habitats yielded in this study are not detailed, and possibilities are not excluded that the derived ribbon-like rooted plants may occur in microhabitats different from those of the foliose plants in the same habitats and that each root form has both major advantages and minor disadvantages, with which the two root forms survive. Further studies are necessary, such as fitness analysis using experimental culture sets and an evo-devo study of the root forms.