Development of conceptacles in Amphiroa ( Corallinales , Rhodophyta )

Here, we describe the development of sporangial and gametangial conceptacles for Amphiroa beauvoisii and A. vanbosseae; sporangial conceptacles only for A. misakiensis; and gametangial conceptacles only for A. cryptarthrodia and A. rigida. The descriptions are based on the observation of histological preparations obtained from 112 specimens collected from the Gulf of California, in Mexico, and the Azores archipelago of Portugal. Information on the development of the sporangial conceptacle pore and conceptacle senescence is here described and illustrated for the first time. Four development patterns were observed: two for sporangial conceptacles; one for spermatangial conceptacles; and one for carposporangial conceptacles. The phases of development of the sporangial conceptacle were found to be useful in delimiting species within the genus. Based on the sporangium location on the cavity floor and the pore canal anatomy, the species A. beauvoisii, A. misakiensis and A. vanbosseae can be distinguished from each other.


Introduction
Information concerning the reproduction of articulated coralline algae has been provided by several authors, thus expanding knowledge of the Corallinaceae family.Nevertheless, the reproductive structures are still poorly described for many genera of articulated coralline algae.In general, all members of the Corallinaceae produce sporangia, carposporangia and spermatangia within uniporate conceptacles, whose development undergoes a number of stages (Johansen 1968).Although the transformation from vegetative to fertile tissue is not fully understood in the Corallinaceae, it is known to involve the local destruction of cells, which, when combined with continued accelerated growth of surrounding tissue, results in the depression of peripheral cells (Johansen 1972), a process that differs among genera (Johansen 1968).Historically, the morphology and anatomy of conceptacles, together with other features, have been used to distinguish genera within the geniculate Corallinaceae.
A description of the genus Amphiroa, encompassing a summary of the diagnostic characteristics, is provided by Womersley (1996) and Harvey et al. (2009).Johansen (1972), in a revision of Corallinaceae conceptacles, described a general pattern for the development of tetrasporangial conceptacles in Amphiroa in which the roof is formed by intersporangial growth and the chamber is opened via a single pore.Additional information on the development of sporangial and gametangial/carposporangial conceptacles was published by Johansen (1968), Murata & Masaki (1978) and Srimanobhas & Masaki (1987).Several other taxonomic studies have included general information on the developmental phases of Amphiroa (Ganesan 1968;Norris & Johansen 1981;Riosmena-Rodríguez & Siqueiros-Beltrones 1991, 1996;Womersley 1996;Moura & Guimarães 2002;Harvey et al. 2009).Aspects of the reproduction of a few species within Amphiroa have been described: by Segawa (1940a,b) for A. rigida J.V. Lamouroux and A. misakiensis Yendo; by Johansen (1968) for A. ephedraea (Lamarck) Decaisne; by Murata & Masaki (1978) for A. zonata Yendo; and by Srimanobhas & Masaki (1987) for A. itonoi V. Srimanobhas & T. Masaki.As a result, different types/patterns of development have been described for Amphiroa species.A sporangial origin has been recorded in the center/periphery (Johansen 1968;Harvey et al. 2009) or in the center and periphery separately (Ganesan 1971;Segawa 1940b;Srimanobhas & Masaki 1987).Two types of development have been described for the sporangial pore canal anatomy, one in which there is a ring of large block-shaped cells (Norris & Johansen 1981; Acta bot.bras.27(4): 698-708.2013.Harvey et al. 2009) and another in which there are no block--shaped cells (Johansen 1968).Two patterns have also been described for the origin of the carposporangial filaments in the fusion cell: from the margins (Ganesan 1968;Riosmena--Rodríguez & Siqueiros-Beltrones 1996) or from the entire surface (Ganesan 1971).Despite all of these data, there is as yet no agreement across studies.For example, the origin of the carposporangial filaments in the fusion cell A. rigida is reported to be from the margins (Riosmena-Rodríguez & Siqueiros-Beltrones 1996) and from the entire surface (Segawa 1940b).This indicates the need for further studies on the development of the reproductive structures within Amphiroa.
In this study, we describe the patterns of development for sporangial and gametangial conceptacles of Amphiroa beauvoisii J.V. Lamouroux and A. vanbosseae Me.Lemoine; sporangial conceptacles of A. misakiensis; and gametangial conceptacles for A. cryptarthrodia Zanardini and A. rigida.Furthermore, comparisons are made among species and with published information and the taxonomic value of conceptacle development for species segregation is discussed.

Material and methods
A total of 112 specimens were studied, including fresh specimens and historical collections: sporangial/gametangial plants of Amphiroa beauvoisii J.V. Lamouroux (40 specimens) and A. vanbosseae Me.Lemoine (44 specimens); sporangial plants of A. misakiensis Yendo (10 specimens); and gametangial plants of A. cryptarthrodia Zanardini (10 specimens) and A. rigida J.V. Lamouroux (eight specimens).Fresh specimens were collected intertidally and subtidally, at a depth of 30 m, at 18 locations; 10 in the Gulf of California, in Mexico, three in the northeastern Mexican Pacific (Fig. 1) and five in the Azores archipelago of Portugal (Fig. 2).Collecting sites and voucher specimens are listed in Tab. 1. Algae were removed from the substrate with a hammer and chisel, brought to the laboratory and fixed in 4% formalin (v/v) in seawater.Specimens were stored in the Phycological Herbarium of the Autonomous University of Baja California Sur, in Baja California Sur, Mexico (code, FBCS) and in the Ruy Telles Palhinha Herbarium of the University of the Azores, in Ponta Delgada, Portugal (code, AZB).Histori cal collections encompass dried specimens of A. cryptarthrodia and A. beauvoisii from the Dutch oceanographic CANCAP V expedition housed at the National Herbarium Nederland, Leiden University branch, in Leiden, the Netherlands (code, L).Herbarium abbreviations are as in Holmgren & Holmgren (1998).

Sporangial conceptacle development
We investigated the development of the sporangial conceptacles prior to conceptacle pore formation in three species (Amphiroa beauvoisii, A. misakiensis and A. vanbosseae) and found it to be identical all three.Therefore, the various stages are illustrated by selected examples of these taxa.The youngest stage observed is the formation of a conceptacle is the appearance of an initial conceptacle dome with a cellular cap (Johansen 1968).A group of peripheral cells in a subsurface stratum elongates anticlinally; these cells are designated cavity cells (Johansen 1968), as shown in Fig. 3 and 4. The cavity cells elongate and divide to form rows of short overlying cells (Fig. 5).These layers of small cells, derived from the cavity cells, are pushed upward by further elongation of the cavity cells and eventually become the conceptacle roof (Fig. 6).Before cavity cells reach their full length, the destruction of some of them begins.Atrophy of the cavity cells forms a cavity, the so-called conceptacle chamber, followed by the development of the reproductive structures.
For all three of the species evaluated (Amphiroa beauvoisii, A. misakiensis and A. vanbosseae), atrophy of the cavity cells creates space for the sporangial initials (Fig. 7).Sporangial initials (Fig. 8-15) develop in the periphery of the chamber floor (A.beauvoisii, Fig. 8) or in the periphery and center (A.misakiensis and A. vanbosseae, Fig. 12 and  14).In all three species, while the initials are still very small, they elongate and divide transversally to form the sporangia mother cells and their stalk cells (Fig. 11 and 13).Autolysis continues until the adjacent cavity cells are largely destroyed.Further growth mostly involves tetrasporangial or bisporangial maturation (Fig. 14 and 15, respectively) and cavity cell destruction.An exception occurs in A. beauvoisii (Fig. 8, 10 and 15), in which cavity cells are still present in conceptacles with mature sporangia.
The conceptacle pore (Fig. 16-22) is initiated by an increase in the volume of subepithallial cells in the central region of the roof cavity (Fig. 16), to form large block-shaped cells (Fig. 19).The cells in the roof and immediately below the block-shaped cells elongate centripetally and migrate towards the future location of the pore (Fig. 17 and 18), compressing the cavity cells.Their continuous elongation causes the autolysis of the large block-shaped cells and the formation of the conceptacle pore (Fig. 19).A different pattern occurs in Amphiroa misakiensis, in which no large--block shaped cells appear.Instead, the cells located in the center of the roof elongate (Fig. 20) and those closest to the cavity show signs of atrophy (Fig. 21).The decalcification and atrophy of the cells immediately below the subepithallial cells results in the formation of a canal leading to the exterior (Fig. 22).This canal soon becomes lined with small, centripetally oriented elongated roof cells.
The final stage of development observed was senescence of the conceptacle.Two patterns were observed; the first, observed in the sporangial conceptacles of Amphiroa misakiensis (Fig. 23 and 24), was initiated by the downward, lateral and upward growth of the cells of the conceptacle floor and the second pattern, observed in the sporangial conceptacles of A. beauvoisii and A. vanbosseae, was initiated solely by the downward and lateral growth of the cells of the conceptacle floor (Fig. 25 and 26).For all conceptacle types, senescence occurred in the presence of tetrasporangia (Fig. 26).

Gametangial and carposporangial conceptacle development
We evaluated the development of male and female conceptacles in A. beauvoisii, A. cryptarthrodia, A. rigida and A. vanbosseae.Because conceptacle development was identical in all of those species, the various stages are illustrated with examples selected from among those taxa.The first stage in the development of male and female conceptacles is characterized by the anticlinal elongation and division of a group of peripheral cells in a subsurface stratum of the intergenicula.Those cells are designated cavity cells (Johansen 1968).The continued elongation and delayed cell division result in the formation of an initial conceptacle dome with a cellular cap (Johansen 1968), as shown in Fig. 27.The cavity cells secrete a mucilaginous material from their distal portion, the cap, formed by copious amounts of light, clear material (Fig. 27).Due to the presence of the cap, the epithallial cells and the central peripheral cells of the dome become detached (Fig. 28).
Cavity cell dissolution begins above the male gametangial initials and progresses upward toward the cavity cells and the conceptacle dome (Fig. 28).Following this destruction, the surrounding tissue grows over the fertile layer, forming the conceptacle roof (Fig. 29 and 30).Because the reproductive initials are still relatively small when this growth occurs, the lack of filling material within the cavity allows the roof to   grow downward as well as centripetally.The terminal part of the roof elongates and divides transversely (Fig. 31), providing new cells that repeat the process until the pore canal formation is completed.In the female gametangial initials, the process follows the same pattern, with cavity cell dissolution (Fig. 32 and 33), followed by the formation of a conceptacle roof and pore canal (Fig. 33 and 34).Male gametangia were observed only for Amphiroa beauvoisii and A. vanbosseae.Their development begins with the formation of basal cells from the transversal basal division of the cavity cells (Fig. 35).After cavity cell dilution, each basal cell forms two spermatangial mother cells and the spermatangia continuously derive from the spermatangia mother cells  to form simple and unbranched filaments.The spermatangia in turn, liberate spermatia that fill the chamber (Fig. 38).
Female gametangia were observed for Amphiroa beauvoisii, A. cryptarthrodia, A. rigida and A. vanbosseae.Their development initiates with the elongation and transverse division of the basal region of the central cavity cells (Fig. 39).As a consequence, three new layers of cells cover the central region of the cavity floor; the elongation of cells from the terminal layer gives rise to the procarp initials (Fig. 39).Each of these, by elongation and transverse division, leads to the formation of a supporting cell and carpogonia branch initial (Fig. 40).The elongation and further division of the latter form the trichogyne (Fig. 41 and 42).The fact that the basic reproductive features are similar to those in other coralline red algae (Lebednik 1977) suggests that spermatia fuse with trichogynes and that male and female haploid nuclei then combine to form a diploid nucleus.The development of carposporangial filaments was observed for Amphiroa cryptarthrodia, A. rigida and A. vanbosseae.This phase initiated when the trichogynes were well developed (Fig. 43).The presence of a carpogonial branch and a discontinuous flat disc-like fusion cell, supporting a few residual carpogonia on its dorsal part (Fig. 44 and 45), provided evidence that fertilization had occurred.In A. vanbosseae, the carposporangial filaments arose solely from the margins of the fusion cell (Fig. 46), whereas in A. cryp-tarthrodia and A. rigida, they arose over the entire surface of the fusion cell (Fig. 47).The cells of the carposporangial filaments increase in size, the upper being relatively large and constituting the carposporangium (Fig. 48).
Two patterns were observed for the senescence of gametangial/carposporangial conceptacles.The first, observed in carposporangial conceptacles (Fig. 49 and 50), was initiated by the downward, lateral and upward growth of the cells of the conceptacle floor.The second pattern, observed in the gametangial conceptacles (Fig. 51 and 52), was initiated only by downward and lateral growth.For all conceptacle types, senescence occurred in the presence of carposporangia (Fig. 49) and gametangia (Fig. 51).between species within Amphiroa, the most diverse geniculate genus of the subfamily Lithophylloideae.
The development of the conceptacle pore is described and illustrated here for the first time, as is conceptacle senescence, for which four patterns were observed: two for sporangial conceptacles, one for spermatangial conceptacles and one for carposporangial conceptacles.As first described for Amphiroa by Johansen (1972), Murata & Masaki (1978) and Srimanhobas & Masaki (1987), we confirmed that conceptacle roof development was by intersporangial growth for sporangial conceptacles and by vegetative filament growth up and over the chamber for the gametangial conceptacles.The development of the sporangial conceptacle cavity by elongation and atrophy of cavity cells described for the genus by Johansen (1972) was also confirmed.Nevertheless, the presence of intact cavity cells among mature sporangia described and illustrated for Amphiroa zonata by Murata & Masaki (1978) was observed only for A. beauvoisii (cf.Fig. 15).The presence of cavity cells in mature sporangial conceptacles in this species was previously reported by Harvey et al. (2009).
The development of the carposporangial filament, previously suggested to be important for species identification within the genus Fosliella (Chamberlain 1977), was also found to be important for the genus Amphiroa.In A. cryptarthrodia and A. rigida, we found that the carposporangial filament arose from the surface of the fusion cell.This finding was previously reported for A. rigida by Segawa (1940b); for A. foliacea by Ganesan (1968); for A. zonata for Murata & Masaki (1978); and for A. beauvoisii and A. valonioides by Riosmena-Rodríguez & Siqueiros-Beltrones (1996).It is of note that Riosmena--Rodríguez & Siqueiros-Beltrones (1996) reported a different pattern for A. rigida from the Gulf of California.In A. vanbosseae, we found that the carposporangial filament arose only from the margins of the fusion cell, as previously reported by Riosmena-Rodríguez & Siqueiros--Beltrones (1996) and Moura & Guimarães (2002) and by Riosmena-Rodríguez & Siqueiros-Beltrones (1996) for A. misakiensis (Tab.2).
We confirmed the diagnostic importance of the sporangial pore canal anatomy in distinguishing between species within the subfamily Lithophylloideae, namely in Lithophyllum (Woelkerling & Campbell 1992;Keats 1997;Harvey et al. 2005) and Amphiroa (Harvey et al. 2009).The type of sporangial pore canal anatomy reported in this study for Amphiroa beauvoisii and A. vanbosseae was also previously reported by Harvey et al. (2009), Norris & Johansen (1981) and Ganesan (1971).However, the present study reports and illustrates, for the first time, the complete occlusion of the pore canal by large block-shaped cells in both species (Fig. 8).
Based on the pore canal anatomy and the location of sporangia on the cavity floor, we created the following key, by which three of the five studied Amphiroa species can easily be distinguished:

Discussion
The present study describes the conceptacle development of Amphiroa species.Our findings clearly demonstrate the importance of conceptacle anatomy in distinguishing

Table 1 .
Sample collection information for the studied species of Amphiroa from the Gulf of California, the Northeastern Mexican Pacific and the Azores archipelago.
*Phycological Herbarium of the Autonomous University of Baja California Sur (code, FBCS).**Ruy Telles Palhinha Herbarium of the University of the Azores (code, AZB).