Origin and evolution of Petrocosmea (Gesneriaceae) inferred from both DNA sequence and novel findings in morphology with a test of morphology-based hypotheses

Analyses of DNA sequence and morphological data separately

The combined cpDNA matrix, which comprises six chloroplast regions of trnL-F, matK, rps16, atpI-atpH, trnH-psbA, and trnT-L, had aligned sequences of 5662 bp, of which 4719 (83.35 %) were constant, 560 (9.89 %)
were variable but uninformative, and 383 (6.76 %) were parsimony informative. We were
unable to amplify cpDNA regions from P. confluens. Modeltest indicated GTR?+?G as the best-fit model for the cpDNA sequence data. The
strict consensus of 6 trees yielded by MP (Maximum Parsimony) analysis (L?=?1182,
CI?=?0.884, RI?=?0.873) was generally congruent with the ML (Maximum Likelihood) tree
and the majority rule BI (Bayesian Inference) tree in the topology (Additional file
1: Figure S2). Support values less than 50 % are marked with asterisk.

In the nuclear DNA analysis with P. confluens added to the matrix, the ILD (incongruence length different) test gave a p value
of 0.42, indicating that the sequence data from ITS and PeCYC1D were congruent. The combined nuclear DNA matrix of ITS and PeCYC1D consisted of 1662 bp, of which 1213 (72.98 %) were constant, 228 (13.72 %) were variable
but uninformative, and 221 (13.3 %) were parsimony informative. Modeltest indicated
GTR?+?G as the best-fit model for the combined nuclear DNA data. The strict consensus
of eight trees from MP analysis (L?=?642, CI?=?0.872, RI?=?0.849) was congruent with
the ML tree and the majority rule consensus BI tree (Additional file 1: Figure S3).

In the combined cpDNA and nuclear DNA analysis, P. rosettifolia and P. longianthera were removed because of their obvious topological differences between cpDNA and nuclear
DNA data, but P. confluens was included despite lacking cpDNA data. The ILD test gave a value of p?=?0.25, indicating
that the data from the two distinct genome regions excluding these two species did
not contain significant incongruence. Modeltest suggested that the GTR?+?G model best
fit the combined data. The combined datasets consisted of 7320 bp, 774 (10.57 %) of
which were variable and 587 (8.02 %) parsimony informative sites. Parsimony analyses
resulted in a single tree (L?=?1767, CI?=?0.886, RI?=?0.872) which was congruent with
the ML tree and the majority rule consensus BI tree (Fig. 2).

Fig. 2. The majority rule consensus Bayesian tree generated from analysis of combined cpDNA
and nDNA data. Bootstrap values from MP/ML are shown above branches and posterior
probabilities from BI are shown below branches. P. Petrocosmea, R. Raphiocarpus, Str. Streptocarpus

The MP-ML-BI tree of the combined cpDNA and nuclear DNA datasets was similar to the
cpDNA and nuclear DNA trees but with stronger support (Figs. 2, Additional file 1: Figure S2-S3). The combined cpDNA and nuclear DNA tree comprises five main clades
labeled A–E (Fig. 2). Each clade receives strong or maximum support, and they are grouped together successively
by strong to maximum support (Fig. 2).

For the analysis of the morphological data, Forty-one morphological characters were
coded. The strict consensus of 125 trees yielded from the MP analysis (L?=?82, CI?=?0.842,
RI?=?0.972) was congruent with the majority rule consensus BI tree (Additional file
1: Figure S4). Similar to the DNA trees, the morphological tree comprises five major
clades including the same species as the molecular based trees. However, most nodes
within the major five clades have weak to moderate support with frequent polytomies.

Analysis of combined DNA sequence and morphological data

In the analysis of the combined data of DNA and morphology with P. rosettifolia and P. longianthera removed, the ILD test gave a value of p?=?0.082, indicating that the data from the
DNA and morphological data did not contain significant incongruence. Both P. rosettifolia and P. longianthera were removed from the combined molecular and morphological analyses due to the discrepancies
in the placement of these two species with ITS and cpDNA. The combined data sets consisted
of 7361 bp, 774 (10.51 %) of which were variable and 628 (8.53 %) parsimony informative
sites. Parsimony analyses resulted in a single tree (L?=?1853, CI?=?0.882, RI?=?0.888)
which was congruent with the majority rule consensus BI tree (Fig. 3).

Fig. 3. Single most parsimonious trees generated from analysis of combined DNA and morphological
data. Note. Bootstrap values from MP are shown above branches and posterior probabilities
from BI are shown below branches. P. Petrocosmea, R. Raphiocarpus

The trees of the combined data set of DNA and morphology and the combined DNA data
are identical in topology with only a few fluctuations in support values of some branches
(Figs. 2-3). The tree of combined DNA and morphological data consists of five major clades labeled
A-E with strong to maximum support, which are clustered together with maximum support
(Fig. 3). Clade A, which consists of four taxa (P. kerrii var. kerrii, P. kerrii var. crinita, P. menglianensis, and P. grandifolia) of sect. Deinanthera sensu Wang (1985) 9] and one species (P. parryorum) of sect. Anisochilus sensu Wang (1985) 9], is sister to the remaining species with maximum support. The five species bear a
series of synapomorphies exclusive to clade A, i.e., vestigial caulescent habit with
ascendant leaves, an upper lip slightly shorter than the lower lip in length, anthers
that are constricted at the tip and two dark red-brown spots on the lower side of
the corolla-tube below the filaments (Figs. 1, 4). In addition, P. kerrii var. kerrii is sister to P. parryorum with maximum support, a relationship that is morphologically reflected in the shared feature of blue-violet
flowers with geniculate filaments. In contrast, P. kerrii var. crinita is sister to P. grandifolia/P. menglianensis with maximum support rather than sister to the type variety of P. kerrii, consistent with their shared traits of white flowers with straight filaments. Petrocosmea kerrii var. kerrii and P. kerrii var. crinita are apparently two independent species because they are not recovered as an exclusive
monophyletic group.

Fig. 4. Photos of dissected flowers of representative species of different clades. 1-3 (clade
A, P. grandifolia): 1.longitudinal section, showing relative position of stamen and pistil inside corolla
tube, 2. anthers, showing anthers constricted at top, poricidal with short filament,
3. pistil, showing style’s tip curving downward, Ca showing corolla throat ribbed
at both upper and lower sides and relative position of style at throat magnified 1.7
times in size relative to Fig. 1-2. Scale bars?=?2.7 mm (1), 1.4 mm (2) and 2.2 mm
(3) (3.5 mm in Ca). 4-6 (clade B, P. mairei var. intraglabra): 4. longitudinal section, 5. Stamen, showing poricidal anther basifixed with straight
filament (5’. anther of P. coerulea showing dehiscent pore), 6. Pistil, Cb showing corolla throat of P. coerulea ribbed at upper side and relative position of style at throat magnified 2.5 times
in size relative to Fig. 1-8. Scale bars?=?3.4 mm (4), 1.1 mm (5) (0.86 mm in 5’)
and 1.3 mm (6) (2 mm in Cb). 7-9 (clade C, P. sericea): 7. longitudinal section, 8. Stamen, showing poricidal anther basifixed with long
geniculate filament (8’. anther of P. minor showing dehiscent pore), 9. pistil, showing style curving downward at top, Cc showing
corolla throat unribbed and relative position of style at throat magnified 2 times
in size relative to Fig. 1-13. Scale bars?=?2.9 mm (7), 1.8 mm (8) (1.6 mm in 8’)
and 2.2 mm (9) (2.1 mm in Cc). 10-12 (clade D, P. forrestii and P. barbata): 10. longitudinal section of P. barbata, showing style extending from centre of the throat, 11. Stamen of P. forrestii, showing anther longitudinal dehiscent with short filament, 12. Pistil of P. forrestii, erect, Cd showing corolla throat of P. barbata unribbed and relative position of style at throat magnified 1.75 times in size relative
to Fig. 1-17. Scale bars?=?3.6 mm (10), 1.3 mm (11) and 1.9 mm (12) (2.6 mm in Cd).
13-15 (clade E, P. sinensis): 13. longitudinal section, 14. Stamen, showing longitudinal anther with short filament
(14’. showing anther with longitudinal dehiscence becoming visible), 15. pistil, showing
style curving downward at the base and curving upward at the top, Ce showing corolla
throat unribbed and relative position of style at throat magnified 1.26 times in size
relative to Fig. 1-24. Scale bars?=?3.7 mm (13), 1.4 mm (14 and 14’) and 2.9 mm (15)
(4.2 mm in Ce). CT, constriction; G, geniculate; L, lower lip; P. dehiscent pore;
U, upper lip

Clade B contains eight taxa (P. coerulea, P. begoniifolia, P. melanophthalma, P. confluens, P. hexiensis, P. duclouxii, P. sichuanensis, and P. mairei var. intraglabra) of sect. Anisochilus sensu Wang (1985) 9], and is a well-supported clade sister to clades C-D with maximum support. Petrocosmea mairei var. intraglabra and P. sichuanensis as a pair of sister species with maximum support are strongly supported to come together
successively with P. duclouxii (MP-BS (bootstrap) =96 %; PP (posterior probabilities) =100 %), P. hexiensis (MP-BS?=?99 %; PP =100 %), and P. confluens (MP-BS?=?98 %; PP?=?100 %). Petrocosmea coerulea and P. melanophthalma as sister species with moderate support (MP-BS?=?78 %; PP?=?98 %) are further clustered
together with P. begoniifolia with MP-BS?=?70 % and PP?=?100 %). The two branches in clade B are further joined
together with strong support (MP-BS?=?97 %; PP?=?100 %). The species of clade B are
defined by their short upper lips with semiorbicular corolla lobes. The morphological
synapomorphies of clade B also include two upper corolla lobes highly reflexed backward
with two purple spots on the lower side of the corolla-tube below the filaments (Fig. 1). Apparently, P. mairei var. intraglabra is a species apart from P. mairei var. mairei which is nested in clade D (Figs. 2-3).

Clade C includes eight taxa (P. iodioides, P. martinii var. leiandra, P. martinii var. martinii, P. minor, P. sericea, P. shilinensis, P. xingyiensis and P. huanjiangensis) of sect. Anisochilus and two species (P. grandiflora and P. yanshanensis) of sect. Petrocosmea. There are two lineages in Clade C with maximum support. In one lineage, P. grandiflora and P. yanshanensis as strongly supported sister species (MP-BS?=?97 %; PP?=?100 %) are grouped in sequence
with P. sericea (MP-BS?=?98 %; PP?=?100 %), P. martinii var. martini (MP-BS?=?99 %; PP?=?100 %), and maximally supported sister species of P. iodioides and P. martinii var. leiandra. In another lineage, P. minor and P. shilinensis are sister to each other (MP-BS?=?71 %; PP?=?97 %), and further grouped with P. xingyiensis by moderate support (MP-BS?=?73 %; PP?=?100 %), and together they are sister to P. huanjiangensis with strong support (MP-BS?=?98 %; PP?=?100 %).

The eight species traditionally placed in sect. Anisochilus all share a specific floral character; the two upper corolla lobes are fused nearly
their entire length and each lobe is folded and rolled laterally to form a carinate-plicate
shape of the upper lip that encloses the style. In the traditional classification,
the upper lip of these species is only described by the phrase “indistinctly 2-lobed,
emarginate, or undivided”. This specific structure of the upper lip is first recognized
herein in Petrocosmea (Fig. 1). Petrocosmea grandiflora and P. yanshanensis as a pair of sister species exhibit a series of floral characters distinctively different
from other species of clade C (Fig. 5). These two species have striking similarities to species of clade E in the external
appearance of the corolla (Fig. 5), the reason that they all had been formerly placed in sect. Petrocosmea. Nevertheless, the highly fused upper lips in the flowers of P. grandiflora and P. yanshanensis as the synapomorphy shared with other species of clade C hint at membership in clade
C. The similarity between these two species and members of clade E is likely the result
of floral convergent evolution. Clade C is sister to clades D and E with maximum support.

Fig. 5. Photos of flowers of P. yanshanensis, P. rosettifolia and P. longianthera. 1-3. P. yanshanensis: face view (1), lateral view (2) and stamens (3); 4-6. P. rosettifolia: face view (4), lateral view (5) and stamen indicating poricidal anther (6); 7-9.
P. longianthera: face view (7), lateral view (8) and stamens indicating long anthers with short filaments
(9). Scale bars?=?5.7 mm (1-2), 1.4 mm (3), 6 mm (4), 5.6 mm (5), 1.6 mm (6), 5.4 mm
(7-8) and 1.8 mm (9). L, lower lip; P, dehiscent pore; U, upper lip

Clade D comprises six taxa (P. forrestii, P. mairei var. mairei, P. barbata, P. cavaleriei, P. xanthomaculata, and P. longipedicellata) of sect. Anisochilus and two newly described species P. nanchuanensis and P. glabristoma with strong support (MP-BS?=?98 %; PP?=?100 %). Petrocosmea nanchuanensis is sister to a maximally supported branch containing P. barbata, and P. longipedicellata gathered together by strong support (MP-BS?=?91 %; PP?=?100 %) with two maximally
supported sister species, P. cavaleriei and P. xanthomaculata. These five species as a maximum supported branch are further united with three well
resolved sister species P. glabristoma, P. forrestii and P. mairei var. mairei. The species in clade D have a generally similar bilateral corolla to the species
in clade B. However, the two lobes in the upper lip are extended forward rather than
reflexed backward. In addition, they can also be easily recognized by two bright yellow
spots or cicatrices on the lower lip and hairs on the upper lip in the corolla throat
(Fig. 1).

Five species (P. nervosa, P. oblata, P. flaccida, P. sinensis, and P. qinlingensis) of sect. Petrocosmea form clade E with maximum support. In clade E, P. oblata and P. flaccida are sister with maximum support and these two are grouped with another set of sister
species, P. sinensis and P. qinlingensis, with strong support (MP-BS?=?90 %; PP?=?100 %). Petrocosmea nervosa is sister to the remaining species in Clade E with maximum support. The species of
clade E all share a large bilobed upper lip that is equal or almost equal to the trilobed
lower lip (Fig. 1). Correspondingly, their styles are generally located in the center of the flower.
In addition, the longitudinal anthers, and three yellow spots on the upper side of
the corolla tube below the filaments are unique to the species of clades D and E,
supporting their sister relationship.

Ancestral area and character state reconstructions

The results of ancestral area reconstruction using S-DIVA in RASP is shown in Fig. 6. The most recent common ancestor of Petrocosmea is in the border region of China, Thailand, India, and Myanmar, lying east and southeast
of Himalaya-Tibetan Plateau. Petrocosmea has greatly diversified in southwestern China, especially in Hengduan Mountain-Yungui
Plateau region, and further spread to central China (Fig. 6).

Fig. 6. Geographical distribution and ancestral area reconstruction of Petrocosmea based on the combined cpDNA and nDNA data. Four areas are defined as follows: Region
A, the border region of China, Thailand, India, and Myanmar, lying east and southeast
of Himalaya Mountain-Tibetan Plateau; Region B, the Hengduan Mountain-Yunnan Plateau
region in southwestern China; Region C, The central China; Region D, the north-central
China (only one species, i.e. Petrocosmea qinlingensis, belonging to clade E, is distributed in Qinling Mountains (Shanxi province) in north-central
China)

For ancestral character state reconstructions, twelve diagnostic characters were analyzed
on the posterior set of trees derived from the combined molecular data analysis (Fig. 2). These were selected among all of the characters that were scored because they may
represent important adaptations in the speciation of Petrocosmea. They are plant habit, ratio of the upper lip to lower lip, structure of the upper
lip; character of corolla throat, dorsoventrally equal/unequal development of the
ovary, length ratio of corolla tube to corolla lobes, inflation of the lower part
of the corolla tube, position of the anther and filament relative to the ovary and
style, type of anther dehiscence, exsertion of the style with curvature type of style
tip; constriction at the top of the anther and straight/geniculation of filaments
(Figs. 1, 4, 7-8, Additional file 1: Figure S5). We found that the plants of clade A retained a vestigial caulescent
habit with ascendant leaves, which transitioned to a habit consisting of a short rhizome
with rosette leaves spreading on the ground (Fig. 1). A ratio of upper to lower lip of 1:2 was inferred to have appeared independently
two times in clades B and D. The upper lip is reflexed backward in clade B but extended
forward in clade D (Figs. 1, 7). The upper to lower lip ratio is 1:4 in the main branch of clade C, but secondarily
lengthened to equal length of the lower lip in clade E as well as the P. grandiflora/P. yanshanensis branch of clade C (Figs. 1, 5, 7). Corolla throat ribbing and whether the gynoecium develops equally or unequally
dorsoventrally were correlated in all taxa and character state mapping indicates that
a corolla throat that is ribbed on both upper and lower surfaces and a gynoecium that
develops only slightly unequally dorsoventrally is the ancestral state for Petrocosmea (Fig. 8). Similarly four other characters were correlated; corolla tube length, corolla tube
inflation on lower side, number of fertile stamens and type of dehiscence, and exsertion
and orientation of the style. The ancestral states for these are a corolla tube that
is equal to slightly longer than the lobes, is inflated on the lower surface, two
fertile stamens with poricidal dehiscence, and an exserted style that is bent downward
(Fig. 8). In clades D and E, the tube is shortened and not inflated and although there are
also only two fertile stamens, their dehiscence is longitudinal and the exserted style
is bent upward (Fig. 8).

Fig. 7. Reconstruction of ancestral states for three morphological characters using Mesquite.
Note: An asterisk in the P. grandiflora/P. yanshanensis branch in clade C indicates a long upper lip but lobed to 1/4 or 1/3 that is distinctive
from clade E (B)

Fig. 8. Reconstruction of ancestral states for seven morphological characters using Mesquite

A series of novel morphological traits are correlated with cladogenetic events in
Petrocosmea. These morphological novelties are mainly reflected in the size and shape of the
upper lip. In clade A, the two upper corolla lobes are slightly smaller than the three
corolla lobes of the lower lip, generating a moderate floral zygomorphy as in Raphiocarpus. In clade B, the two upper corolla lobes are remarkably reduced relative to the three
lobes of the lower lip. In clade C, the two much shortened upper corolla lobes are
fused and extremely specialized. In clade D, even though the upper lobes are in general
similar to those in clade B in size, they are extended forward with a flat face, contrasting
with the two upper corolla lobes reflexed backward in clade B. The flowers in clade
E are nearly actinomorphic, reflected in the equal length of the upper and lower lips,
a deep sinus among the five corolla lobes and a much shortened corolla tube (Fig. 1). These morphological variants in the size and shape of the upper lip are consistent
with a series of counterparts in other floral organs, such as character of corolla
throat, length ratio of corolla tube to corolla lobes, inflation of the lower part
of the corolla tube, position of the anther and filament relative to the ovary and
style and type of anther dehiscence, exsertion of the style with curvature type of
style tip, and dorsoventrally equal/unequal development of the ovary (Figs. 1, 4, 8).