Stem Carbon Dioxide Efﬂux of Lignophytes Exceeds That of Cycads and Arborescent Monocots

: Tree stem CO 2 efﬂux (Es) can be substantial and the factors controlling ecosystem-level Es are required to fully understand the carbon cycle and construct models that predict atmospheric CO 2 dynamics. The majority of Es studies used woody lignophyte trees as the model species. Applying these lignophyte data to represent all tree forms can be inaccurate. The Es of 318 arborescent species was quantiﬁed in a common garden setting and the results were sorted into four stem growth forms: cycads, palms, monocot trees that were not palms, and woody lignophyte trees. The woody trees were comprised of gymnosperm and eudicot species. The Es did not differ among the cycads, palms, and non-palm monocots. Lignophyte trees exhibited Es that was 40% greater than that of the other stem growth forms. The Es of lignophyte gymnosperm trees was similar to that of lignophyte eudicot trees. This extensive species survey indicates that the Es from lignophyte tree species do not align with the Es from other tree growth forms. Use of Es estimates from the literature can be inaccurate for understanding the carbon cycle in tropical forests, which contain numerous non-lignophyte tree species.


Introduction
The efflux of carbon dioxide (CO 2 ) from tree stem surfaces (Es) has been extensively studied to answer various questions and more fully understand the global carbon cycle [1,2]. As with many aspects of biology research, the Es literature is biased toward one subset of biodiversity. Most case studies of tree Es have focused exclusively on lignophyte species with stems comprised mostly of wood constructed by true bifacial secondary cambium. This expansive literature contains only a few examples in which pachycaulous tree species with stems devoid of bifacial secondary cambium were represented [3][4][5][6].
A major contributor to Es is stem tissue respiration. However, numerous interacting factors coalesce to define Es in space and time. For example, CO 2 from root respiration can be transported to stems by way of xylem, and this CO 2 can exit xylem within stems to increase the Es above that of stem tissue respiration [7,8]. This transported CO 2 is under the influence of diel variations in sap flow [9,10]. The movement of CO 2 from the internal tissues to stem surfaces can also be under the control of temporal storage or re-fixation [11]. These and other interacting factors can cause the Es to be heavily influenced by CO 2 that was respired from tissues that are distant from the site of efflux [12].
A recent study designed to understand the diel patterns of Es for arborescent cycads, monocots, and lignophytes [6] included only six species of each growth form. Other studies that compared different stem tissue anatomy and its influence in Es were restricted to lignophyte species [13][14][15]. An extensive survey to compare the Es of trees with disparate stem growth forms has not been conducted to date in a single forest or garden. I hypothesized that Es from an extensive range of tree species would sort into significantly different groups, based on stem design. The objective of this study was to use the large living collection in a common garden setting to compare the Es of four growth forms used to design and construct tree stems.

Materials and Methods
This study was conducted at Nong Nooch Tropical Botanical Garden in Sattahip, Thailand. The location and characteristics of this living collection have been described [6]. The dates of measurements were 8-15 July 2019. In this setting and this time of year, the Es of non-lignophyte trees was not influenced by the time of day, but the lignophyte trees exhibited greater Es during midday [6]. Therefore, the measurements for this extensive species survey were restricted to the hours of 900-1500 h on each day of measurement.
A total of 99 cycad species were included (Table A1). There were 96 lignophyte species included (Table A2). The arborescent monocot species were separated into two groups. A total of 17 arborescent monocot taxa that were not palm species were included (Table A3). Finally, there were 106 palm species in the study (Table A4).
The Es was measured, as previously described [4][5][6]. Vigorous trees with no obvious wounds or decay on the stems were selected. A CIRAS EGM-4 analyzer fitted with a SRC-1 close system chamber (PP Systems, Amesbury, MA, USA) was used to quantify the Es from the stem surfaces. The chamber was secured using modeling clay as the sealant at a stem height of 30-40 cm above the root collar. The EGM-4 recorded the air temperature, and the chamber's increase in CO 2 concentration above ambient was quantified after a 2 min period. The change in CO 2 concentration was used to calculate the flux by dividing by area and time. Three periods of efflux were recorded at different radial locations for each sampling period for each tree.
The stem surface temperature was measured with an infrared thermometer (Milwaukee Model 2267-20, Milwaukee Tool, Brookfield, WI, USA). The relative humidity was determined with a sling psychrometer every hour during the periods of measurements. The stem diameter at the height of measurements and total stem height were measured for each tree.
Two sampling periods were applied to each species. For taxa with more than one large tree, this included two trees. For taxa with a single large tree, the two samples were from the same tree but separated by at least three days. The data were sorted according to four stem growth forms: cycad species, palm species, arborescent non-palm monocot species, and lignophyte species. The data were subjected to ANOVA using the PROC MIXED model (SAS Institute, Cary, NC, USA) with unequal replications. There were 636 observations in the data set, two per species. The two observations were treated as subsamples in the analysis. The means separation was conducted by Tukey's HSD test.

Results and Discussion
The cycad trees were represented by 53 Cycadaceae and 46 Zamiaceae species (Table A1). The stem circumference ranged from 51-169 cm with a mean of 96 cm. The mean stem temperature was 31.8 • C and the concomitant mean air temperature was 32.6 • C. Individual Es measurements ranged from 0.5-6.2 µmol·m −2 ·s −1 . The lignophyte trees were represented by 34 families (Table A2). The stem circumference ranged from 51-156 cm with a mean of 84 cm. The mean stem temperature was 31.3 • C and concomitant mean air temperature was 32.0 • C. Individual Es measurements ranged from 0.2-7.6 µmol·m −2 ·s −1 . The monocot trees that were not palm species were represented by five families (Table A3). The stem circumference ranged from 51-175 cm with a mean of 82 cm. The mean stem temperature was 31.5 • C and the concomitant mean air temperature was 32.1 • C. The individual Es measurements range from 0.8-4.7 µmol·m −2 ·s −1 . The palm species representing the Arecaceae family exhibited a stem circumference ranging from 48-182 cm with a mean of 71 cm (Table A4). The mean stem temperature was 31.7 • C and the concomitant mean air temperature was 32.4 • C. The individual Es measurements ranged from 0.7-7.5 µmol·m −2 ·s −1 . The relative humidity ranged from 56% to 69% and did not change substantially among the hours and dates of the study.
The stem CO 2 efflux differed among the four stem growth forms (F 3,314 = 10.64, p < 0.001). The means separated into two groups, with the lignophyte species exhibiting greater Es than the other three stem growth forms ( Table 1). The lignophyte trees exhibited Es that was 40% greater than the mean of the other growth forms. No differences in the Es occurred among the cycad, palm, and non-palm monocot stem forms. Cycads and monocot trees often produce thick primary growth constructed by a primary thickening meristem, and do not possess bifacial secondary cambium to increase stem diameter at distances away from the stem tip [16][17][18][19][20][21][22]. For all of these trees, the peripheral tissues are ground tissue with vascular tissues embedded closer to the stem center. One of the factors that influences CO 2 efflux from a stem surface is the diffusion and conductance constraints imposed by tissues that are peripheral to tissues that serve as the greatest internal source of CO 2 , such as sap flow in xylem [23]. The substantial radial distance of xylem tissues and other major sources of CO 2 from the stem surface of these pachycaulous trees can account for the greater mean Es for lignophyte trees, which has been shown herein.
Considering the prominence of these pachycaulous trees in tropical forests, the historical exclusion of them from Es studies is unfortunate. Indeed, the CO 2 derived from stem efflux can represent up to 40% of the CO 2 contributed to by vegetation [1,24]. This survey, represented by 222 pachycaulous tree species, confirms the earlier findings based on a limited number of species [6], and indicates that attempts to use the Es literature based on the lignophyte species can over-estimate the Es in regions that are represented by these tree species.
Cycads comprise the most threatened contemporary plant group [25]. Conservation physiology has emerged as a critical component of the suite of conservation strategies, because an understanding of the physiological responses of threatened organisms to their escalating biotic and abiotic threats is required for successful species recovery [26,27]. For federally listed endangered cycad species in the United States, such as Cycas micronesica K.D. Hill (see Table A1), understanding the physiology of the taxa is crucial for developing effective federal recovery plans [28]. Clearly, the pursuit of more cycad physiology studies will advance the nascent discipline of conservation physiology.
Future research on the Es of cycad and monocot trees will be required to fully understand the reasons that mean Es is less than the mean Es of lignophyte trees. The design of cycad stems is fairly homogeneous, with vascular cylinders inserted between the persistent living pith and cortex [29]. The design of palm stems is also fairly homogeneous with vascular bundles scattered through the ground tissue [19,22]. However, the design of the non-palm arborescent monocot tree stems is heterogeneous among the families. A closer look at this group of pachycaulous species can yield interesting findings about what endogenous factors mostly control the Es of these non-lignophyte trees.
In conclusion, the many factors that interact to control the magnitude of CO 2 efflux from tree stem surfaces are differentially expressed among various tree stem designs. The results herein suggest that the traits of stem peripheral tissues can be among the defining factors that cause the differences in Es among various tree growth forms. Acknowledgments: I thank Nong Nooch Tropical Botanical Garden for the logistical support and access to the living collection. I thank Dallas Johnson for the statistical analysis.

Conflicts of Interest:
The author declares no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Appendix A Table A1. List of cycad species included in the carbon dioxide efflux study. Circ = circumference, Air T = air temperature, and Stem T = surface temperature of stems.